Engineered antibody constant domain molecules

ABSTRACT

Described herein are engineered antibody constant domain molecules, such as CH2 or CH3 domain molecules, comprising at least one mutation, or comprising at least one complementarity determining region (CDR), or a functional fragment thereof, engrafted in a loop region of the CH2 domain. The CH2 domain molecules described herein are small, stable, soluble, exhibit little to no toxicity and are capable of binding antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 12/864,758, filed Jul.27, 2010, which is the U.S. National Stage of International ApplicationNo. PCT/US2009/032692, filed Jan. 30, 2009, published in English underPCT Article 21(2), which claims the benefit of U.S. ProvisionalApplication No. 61/063,245, filed Jan. 31, 2008. The above-listedapplications are herein incorporated by reference in their entirety.

FIELD

This relates to antibodies, specifically to antibody constant domainsmutated at specific positions and/or engrafted with one or more variablechain loops from a heterologous antibody that specifically bind anantigen of interest.

BACKGROUND

Conventional antibodies are large multi-subunit protein complexescomprising at least four polypeptide chains, including two light chainsand two heavy chains. The heavy and light chains of antibodies containvariable (V) regions, which bind antigen, and constant (C) regions,which provide structural support and effector functions. The antigenbinding region comprises two separate domains, a heavy chain variabledomain (V_(H)) and a light chain variable domain (V_(L)).Complementarity determining regions (CDRs), short amino acid sequencesin the variable domains of an antibody, provide antigen specificity. Theheavy and light chains of an antibody molecule each provide three CDRs(CDR1, CDR2 and CDR3), therefore there are six CDRs for each antibodythat can come into contact with the antigen, resulting in the antigenspecificity.

A typical antibody, such as an IgG molecule, has a molecular weight ofapproximately 150 kD. Therapeutic use can be limited due to therelatively large size of an antibody, which can restrict tissuepenetration or epitope access.

A number of smaller antigen binding fragments of naturally occurringantibodies have been identified following protease digestion (forexample, Fab, Fab′, and F(ab′)₂). These antibody fragments have amolecular weight ranging from approximately 50 to 100 kD. Recombinantmethods have been used to generate alternative antigen-bindingfragments, termed single chain variable fragments (scFv), which consistof V_(L) and V_(H) joined by a synthetic peptide linker. A scFv moleculehas a molecular weight of approximately 25-30 kD.

While the antigen binding unit of a naturally-occurring antibody inhumans and most other mammals is generally known to be comprised of apair of variable regions, camelid species express a large proportion offully functional, highly specific antibodies that are devoid of lightchain sequences. The camelid heavy chain antibodies exist as homodimersof a single heavy chain, dimerized via their constant regions (U.S. Pat.Nos. 5,840,526 and 6,838,254; and U.S. Patent Application PublicationNo. 2003-0088074). The variable domains of these camelid heavy chainantibodies, referred to as V_(H)H domains, retain the ability, whenisolated as fragments of the V_(H) chain, to bind antigen with highspecificity (Hamers-Casterman et al. Nature 363:446-448, 1993; Gahroudiet al. FEBS Lett. 414:521-526, 1997).

Antigen binding single V_(H) domains, called domain antibodies (dAb),have also been identified from a library of murine V_(H) genes amplifiedfrom genomic DNA of immunized mice (Ward et al. Nature 341:544-546,1989). Human single immunoglobulin variable domain polypeptides capableof binding antigen with high affinity have also been described (see, forexample, PCT Publication Nos. WO 2005/035572 and WO 2003/002609).

However, a need remains for very small antibodies that can specificallybind antigen. Such small molecules could provide increased epitopeaccess, better tissue penetration and could be used for any diagnosticor therapeutic application that utilizes antibodies or their fragments.

SUMMARY

This disclosure concerns engineered antibody constant domain molecules.In one embodiment, the antibody constant domain is a CH2 domain fromIgG, IgA or IgD.

In another embodiment, the antibody constant domain is a CH3 domain fromIgE or IgM. As described herein, the CH2 or CH3 domain molecules aresmall, stable, soluble, have minimal to no toxicity and effectively bindantigen. Thus, provided herein are polypeptides comprising animmunoglobulin CH2 or CH3 domain, wherein at least one of the loops ofthe CH2 or CH3 domains is mutated, or at least a portion of a loopregion of the CH2 or CH3 domain is replaced by a complementaritydetermining region (CDR), or a functional fragment thereof (such as onecontaining specificity-determining residues (SDR)), from a heterologousimmunoglobulin variable domain, or both. The CH2 and CH3 domainmolecules described herein have a molecular weight of less than about 15kD. Also provided herein are compositions, libraries and kits comprisingthe CH2 or CH3 domain molecules, and methods of use. Further providedare recombinant constant domains exhibiting increased stability that canbe used as scaffolds for the construction of antigen binding CH2 or CH3domains. Methods of identifying recombinant CH2 or CH3 domains thatspecifically bind antigen and methods of generating libraries comprisingrecombinant CH2 or CH3 domains are also provided.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic drawing of an immunoglobulin molecule.Conventional antibodies are large multi-subunit protein complexescomprising at least four polypeptide chains, including two light (L)chains and two heavy (H) chains. The heavy and light chains ofantibodies contain variable (V) regions, which bind antigen, andconstant (C) regions (such as CH1, CH2 and CH3 domains), which providestructural support and effector functions. The antigen binding regioncomprises two separate domains, a heavy chain variable domain (V_(H))and a light chain variable domain (V_(L)).

FIG. 1B shows the consensus amino acid sequence of a human heavy chainvariable domain (SEQ ID NO: 1). The locations of CDR1, CDR2, CDR3(denoted H1, H2 and H3) are indicated. Also shown are the amino acidsequences of the heavy chain of three different antigen-specific humanantibodies (SEQ ID NOs: 2-4). The numbers shown are based on the Kabatnumbering system (Wu and Kabat, J. Exp. Med. 132(2):211-250, 1970).

FIG. 2 shows the amino acid sequence of the human β1 CH2 domain (SEQ IDNO: 5). Residues in regions of β-sheet ( . . . ) and α-helices (***) areindicated. The locations of Loop B-C (here denoted as Loop 1), Loop D-E(here denoted as Loop 2), Loop F-G (here denoted as Loop 3), Loop A-B,Loop C-D and Loop E-F are also shown. Residues in each loop are shown inbold.

FIGS. 3A-3C are schematic drawings illustrating potential strategies forgrafting CDRs (or hypervariable loops) on CH2 domains.

FIG. 4 shows an image of a gel demonstrating protein expression ofengineered CH2 domains, which is indicated by the arrow.

FIG. 5A shows an amino acid sequence alignment of human CH2 (NCBAccession No. J00228; SEQ ID NO: 5) and mouse CH2 (NCB Accession No.J00453; SEQ ID NO: 92). Identical and similar residues were 67% and 92%,respectively.

FIG. 5B is a graph showing size exclusion chromatography of human CH2.The inset figure shows the standard curve. FIG. 5C is an image of anSDS-PAGE gel showing the molecular weight of a CH2 domain molecule (atconcentrations of 1-10 μg or 2-5 μg per lane), a single chain variablefragment (scFv), an antibody fragment (Fab) and an intact antibodymolecule (IgG).

FIGS. 6A-6B are graphs showing stability of human CH2 measured bycircular dichroism (CD) and differential scanning calorimetry (DSC). (A)Folding curves at 25° C. (—), unfolding at 90° C. (□□□) and refolding(---) at 25° C. measured by CD. The fraction folded of the protein (ff)was calculated as ff=([θ]−[θ_(M)])/([θ_(T)]−[θ_(M)]). [θ_(T)] and[θ_(M)] where the mean residue ellipticities at 216 of folded state at25° C. and unfolded state of 90° C. Exact T_(m) value (54.1±1.2° C.)from CD was determined from the first derivative [d(Fraction folded)/dT]against temperature (T). (B) Thermo-induced unfolding curve from DSC.T_(m)=55.4° C., which is similar to that from CD.

FIG. 7 is a schematic drawing showing design of m01 and m02 based on theCH2 structure. The distance between two C^(α)s in two native Cys is 6.53Å. These two Cys residues formed a native disulfide bond (indicated byblack arrow). Engineered disulfide bond were introduced between V10 andK104 (m01) or L12 and K104 (m02) replaced by cysteines.

FIG. 8 is an image of an SDS-PAGE gel showing high level of expressionof m01 and m02. Soluble expression of m01 and m02 was compared with thatof CH2. Expression is indicated by the arrows.

FIGS. 9A-9E are graphs showing increased stability of two mutantsmeasured by CD (A-C), DSC (D) and spectrofluorimetry (E). Folding curvesat 25° C. (—), unfolding at 90° C. (□□□) and refolding (---) at 25° C.of m01 (A) and m02 (B) are shown. (C) The fraction folded of m01 and m02was calculated by the same method as for CH2. T_(m) of m01=77.4±1.7° C.,T_(m) of m02=68.6±0.6° C. (D) Thermo-induced unfolding curves of m01 andm02 were also recorded by DSC. T_(m) of m01 and T_(m) of m02 increasedabout 20° C. and 10° C., respectively, compared to CH2. (E) Comparisonof urea-induced unfolding among CH2, m01 and m02 by spectrofluorimetry.The midpoints of unfolding of CH2, m01 and m02 are 4.2, 6.8 and 5.8 M,respectively.

FIG. 10A shows size exclusion chromatography of m01 and m02. As CH2, m01formed only monomer, while m02 primarily formed monomer and to a lesserdegree formed dimer. FIG. 10B is a graph showing high stability ofN-terminally truncated CH2 (CH2s) and truncated m01 (mOls). The firstseven N-terminal residues were deleted (residues 1-7 of SEQ ID NO: 5).The 50% unfolding temperatures (T_(m)s) measured by CD (62° C. and 79°C., respectively) were significantly higher (8° C. and 5° C.,respectively) than those of the corresponding CH2 and m01 (54° C. and74° C., respectively).

FIG. 11 is a schematic showing the design of the CH2 library. Shown is aschematic representation of the CH2 fragment, with filled rectanglesrepresenting the loops (L1-L3). Shaded rectangles represent the Loop (L)and Helixes (H1, H2) facing the opposite direction from loops 1 to 3.Empty rectangles labeled with letters A-G represent the seven β-strandsforming the β sandwich structure. Numbers 231 and 341 represent thestarting and ending residues of the CH2 fragment in the context of theIgG1. Sequences of CH2 loop 1 (SEQ ID NO: 93) and loop 3 (SEQ ID NO: 95)are shown below and underlined. The mutations introduced are shown inbrackets (SEQ ID NOs: 94 and 96).

FIGS. 12A-12B show characterization of the CH2 binders. (A) The four Balgp120-CD4 specific CH2 clones were expressed and purified as describedin the Examples below. The purified product was analyzed by westernblot. Samples 1-4 represent clones m1a1 to m1a3′ from the solublefraction and 5-8 renatured from the inclusion body. (B) ELISA analysisof binding of the CH2 clones to Bal gp120-CD4.

FIGS. 13A-13B are graphs and images of electrophoretic gels showingdeterminants of CH2 specific binding. (A) Loop 1 determines the bindingability. Two of the dominant clones m1a1 and m1a2, as well as the twohybrids containing loop 1 sequences from m1a1 and m1a3 but original CH2loop 3 sequence were expressed and purified from the inclusion body andrefolded (left panel). These proteins were then used in the ELISAanalysis (right panel). (B) CH2 provided critical structural support forloop 1. The dominant clone m1a1 and its mutant carrying an additionaldisulfide bond were expressed, purified and refolded (left panel). Theywere then used in the ELSIA assay (right panel).

FIG. 14 is a graph showing broad neutralization of HIV Env pseudo-typedvirus infection by CH2 binders. The two CH2 clones m1a1 and m1a2, at afixed concentration of 100 μg/ml, were used to test their neutralizingability against a panel of nine HIV pseudoviruses. C34 peptide at aconcentration of 4 or 6 μg/ml was used as the positive control.

FIG. 15 shows the design of the second CH2 library based on m1a1. Loop 2(SEQ ID NO: 97) and Loop 3 (SEQ ID NO: 99) sequences (underlined) fromthe CH2 clone m1a1 were replaced by those shown in parentheses (SEQ IDNOs: 98 and 100).

FIGS. 16A-16D show characterization of CH2 clones selected from thesecond CH2 library. (A) Expression and purification of CH2 clonesselected from the second library. (B) Gel filtration analysis of m1b3.(C) ELSIA analysis of the CH2 clones. (D) The loop 2 and loop 3sequences of the clone m1b3, which had predominantly monomeric form, incomparison to the original CH2 sequences (SEQ ID NOS: 97-100).

FIG. 17 is a graph showing pseudovirus neutralization by clones from thesecond CH2 library. Three clones isolated from the second library wereanalyzed for their neutralizing ability against the same panel of HIVpseudoviruses at a concentration of 100 μg/ml. ScFv X5 purified inparallel was used as a control at a concentration of 20 μg/ml.

FIG. 18 is a graph showing CH2 binder recognized a conserved epitope.The predominantly monomeric CH2 clone m1b3 was biotin labeled and usedin a competition ELISA assay. ELISA antigen Bal gp120-CD4 was coated atthe bottom of the ELISA plate. Fixed amount of biotinylated m1b3 at 1.7μM was mixed with indicated amount of unlabeled m1b3, scFv X5 or m36-Fcand added to each well. The bound m1b3 was detected withstreptavidin-HRP.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Sep. 26, 2013, 45.1 KB, which is incorporated byreference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of a human V_(H) domain.

SEQ ID NOs: 2-4 are the amino acid sequences of the V_(H) domains ofthree human antibodies.

SEQ ID NO: 5 is the amino acid sequence of the human ≡1 CH2 domain.

SEQ ID NOs: 6-10 are nucleotide sequences of PCR primers for generationof a library of mutant CH2 domains.

SEQ ID NOs: 11-30 are the amino acid sequences of fragments of mutantCH2 domains with randomized Loop 1.

SEQ ID NOs: 31-50 are the amino acid sequences of fragments of mutantCH2 domains with randomized Loop 3.

SEQ ID NOs: 51-68 are nucleotide sequences of PCR primers forengraftment of CDR3s from human antibodies into the CH2 scaffold.

SEQ ID NOs: 69-87 are amino acid sequences of fragments of engineeredCH2 domains with grafted H3s.

SEQ ID NOs: 88 and 89 are amino acid sequences of fragments of the CH2domain mutant m01.

SEQ ID NOs: 90 and 91 are amino acid sequences of fragments of the CH2domain mutant m02.

SEQ ID NO: 92 is the amino acid sequence of murine CH2.

SEQ ID NO: 93 is the amino acid sequence of CH2 loop 1.

SEQ ID NO: 94 is the consensus amino acid sequence of mutant CH2 loop 1.

SEQ ID NO: 95 is the amino acid sequence of CH2 loop 3.

SEQ ID NO: 96 is the consensus amino acid sequence of mutant CH2 loop 3.

SEQ ID NO: 97 is the amino acid sequence of CH2 loop 2 from clone m1a1.

SEQ ID NO: 98 is the consensus amino acid sequence of mutant CH2 loop 2derived from clone m1a1.

SEQ ID NO: 99 is the amino acid sequence of CH2 loop 3 from clone m1a1.

SEQ ID NO: 100 is the consensus amino acid sequence of mutant CH2 loop 3derived from clone m1a1.

SEQ ID NOs: 101-105 are the nucleotide sequences of PCR primers foramplification of the first CH2 library.

SEQ ID NO: 106 is the amino acid sequence of an m1a1 synthetic peptide.SEQ ID NO: 107 is the amino acid sequence of m1a1 loop 1.

SEQ ID NO: 108 is the amino acid sequence of m1a2 loop 1.

SEQ ID NO: 109 is the amino acid sequence of m1a3 and m1a3′ loop 1.

SEQ ID NO: 110 is the amino acid sequence of m1a1 loop 3.

SEQ ID NO: 111 is the amino acid sequence of m1a2 loop 3.

SEQ ID NO: 112 is the amino acid sequence of m1a3 loop 3.

SEQ ID NO: 113 is the amino acid sequence of m1a3′ loop 3.

DETAILED DESCRIPTION I. Abbreviations

ADCC: Antibody-dependent cell-mediated cytotoxicity

CDC: Complement-dependent cytotoxicity

CDR: Complementarity determining region

DNA: Deoxyribonucleic acid

ELISA: Enzyme-linked immunosorbent assay

HIV: Human immunodeficiency virus

Ig: Immunoglobulin

NK: Natural killer

RNA: Ribonucleic acid

SDR: Specificity determining residue

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intravenous, thecomposition is administered by introducing the composition into a veinof the subject.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad of immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer.

Each tetramer is composed of two identical pairs of polypeptide chains,each pair having one “light” (about 25 kDa) and one “heavy” (about 50-70kDa) chain. The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The terms “variable light chain” (V_(L)) and “variableheavy chain” (V_(H)) refer, respectively, to these light and heavychains. Each light chain contains a single constant domain (CL), whileeach heavy chain contains three constant domains, CH1, CH2 and CH3 (orfour constant domains for IgE and IgM). See FIG. 1A for a schematicdrawing of a conventional immunoglobulin molecule.

As used herein, the term “antibodies” includes intact immunoglobulins aswell as a number of well-characterized fragments having a molecularweight of about 25 to 100 kD. For instance, Fabs, Fvs, and single-chainFvs (scFvs) that bind to target protein (or an epitope within a proteinor fusion protein) would also be specific binding agents for thatprotein (or epitope). These antibody fragments are defined as follows:(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule produced by digestion of whole antibodywith the enzyme papain to yield an intact light chain and a portion ofone heavy chain; (2) Fab′, the fragment of an antibody molecule obtainedby treating whole antibody with pepsin, followed by reduction, to yieldan intact light chain and a portion of the heavy chain; two Fab′fragments are obtained per antibody molecule; (3) (Fab′)₂, the fragmentof the antibody obtained by treating whole antibody with the enzymepepsin without subsequent reduction; (4) F(ab′)₂, a dimer of two Fab′fragments held together by two disulfide bonds; (5) Fv, a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(6) scFv, single chain antibody, a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Methods of making thesefragments are routine (see, for example, Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies can be monoclonal or polyclonal. Merely by way of example,monoclonal antibodies can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature 256:495-97, 1975)or derivative methods thereof. Detailed procedures for monoclonalantibody production are described, for example, by Harlow and Lane(Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

A “humanized” immunoglobulin, such as a humanized antibody, is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (such as a mouse, rat, or synthetic) immunoglobulin.The non-human immunoglobulin providing the CDRs is termed a “donor,” andthe human immunoglobulin providing the framework is termed an“acceptor.” In one embodiment, all the CDRs are from the donorimmunoglobulin in a humanized immunoglobulin. A “humanized antibody” isan antibody, such as a humanized monoclonal antibody, comprising ahumanized light chain and a humanized heavy chain immunoglobulin. Ahumanized antibody binds to the same or similar antigen as the donorantibody that provides the CDRs. The acceptor framework of a humanizedimmunoglobulin may have a limited number of substitutions by amino acidstaken from the donor framework. Humanized molecules can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. These moleculescan be constructed by means of genetic engineering (for example, seeU.S. Pat. No. 5,585,089).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity.

Autoimmune disease: A disease in which the immune system produces animmune response (for example, a B cell or a T cell response) against anantigen that is part of the normal host (that is, an autoantigen), withconsequent injury to tissues. An autoantigen may be derived from a hostcell, or may be derived from a commensal organism such as themicro-organisms (known as commensal organisms) that normally colonizemucosal surfaces.

Exemplary autoimmune diseases affecting mammals include rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(for example, Crohn's disease, ulcerative colitis), autoimmune gastricatrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes,non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto'sthyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemiclupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and thelike.

Binding affinity: The strength of binding between a binding site and aligand (for example, between an antibody, CH2 domain or CH3 domain andan antigen or epitope). The affinity of a binding site X for a ligand Yis represented by the dissociation constant (K_(d)), which is theconcentration of Y that is required to occupy half of the binding sitesof X present in a solution. A lower (K_(d)) indicates a stronger orhigher-affinity interaction between X and Y and a lower concentration ofligand is needed to occupy the sites. In general, binding affinity canbe affected by the alteration, modification and/or substitution of oneor more amino acids in the epitope recognized by the paratope (portionof the molecule that recognizes the epitope). Binding affinity can bethe affinity of antibody binding an antigen.

In one example, binding affinity is measured by end-point titration inan Ag-ELISA assay. Binding affinity is substantially lowered (ormeasurably reduced) by the modification and/or substitution of one ormore amino acids in the epitope recognized by the antibody paratope ifthe end-point titer of a specific antibody for the modified/substitutedepitope differs by at least 4-fold, such as at least 10-fold, at least100-fold or greater, as compared to the unaltered epitope.

CH2 or CH3 domain molecule: A polypeptide (or nucleic acid encoding apolypeptide) derived from an immunoglobulin CH2 or CH3 domain. Theimmunoglobulin can be IgG, IgA, IgD, IgE or IgM. In one embodimentdescribed herein, the CH2 or CH3 domain molecule comprises at least oneCDR, or functional fragment thereof. The CH2 or CH3 domain molecule canfurther comprise additional amino acid sequence, such as a completehypervariable loop. In another embodiment, the CH2 or CH3 domainmolecules have at least a portion of one or more loop regions replacedwith a CDR, or functional fragment thereof. In some embodimentsdescribed herein, the CH2 or CH3 domains comprise one or more mutationsin a loop region of the molecule. A “loop region” of a CH2 or CH3 domainrefers to the portion of the protein located between regions of β-sheet(for example, each CH2 domain comprises seven β-sheets, A to G, orientedfrom the N- to C-terminus). As shown in FIGS. 3A-3C, a CH2 domaincomprises six loop regions: Loop 1, Loop 2, Loop 3, Loop A-B, Loop C-Dand Loop E-F. Loops A-B, C-D and E-F are located between β-sheets A andB, C and D, and E and F, respectively. Loops 1, 2 and 3 are locatedbetween β-sheets B and C, D and E, and F and G, respectively. See Table1 for the amino acid ranges of the loops in a CH2 domain. The CH2 andCH3 domain molecules disclosed herein can also comprise an N-terminaldeletion, such as a deletion of about 1 to about 7 amino acids. Inparticular examples, the N-terminal deletion is 1, 2, 3, 4, 5, 6 or 7amino acids in length. The CH2 and CH3 domain molecules disclosed hereincan also comprise a C-terminal deletion, such as a deletion of about 1to about 4 amino acids. In particular examples, the C-terminal deletionis 1, 2, 3 or 4 amino acids in length.

CH2 and CH3 domain molecules are small in size, usually less than 15 kD.The CH2 and CH3 domain molecules can vary in size depending on thelength of CDR/hypervariable amino acid sequence inserted in the loopsregions, how many CDRs are inserted and whether another molecule (suchas an effector molecule or label) is conjugated to the CH2 or CH3domain. In some embodiments, the CH2 or CH3 domain molecules do notcomprise additional constant domains (i.e. CH1 or another CH2 or CH3domain) or variable domains. In one embodiment, the CH2 domain is fromIgG, IgA or IgD. In another embodiment, the constant domain is a CH3domain from IgE or IgM, which is homologous to the CH2 domains of IgG,IgA or IgD.

The CH2 and CH3 domain molecules provided herein can be glycosylated orunglycosylated. For example, a recombinant CH2 or CH3 domain can beexpressed in an appropriate mammalian cell to allow glycosylation of themolecule.

Complementarity determining region (CDR): A short amino acid sequencefound in the variable domains of antigen receptor (such asimmunoglobulin and T cell receptor) proteins that provides the receptorwith contact sites for antigen and its specificity for a particularantigen. Each polypeptide chain of an antigen receptor contains threeCDRs (CDR1, CDR2 and CDR3). Antigen receptors are typically composed oftwo polypeptide chains (a heavy chain and a light chain), thereforethere are six CDRs for each antigen receptor that can come into contactwith the antigen. Since most sequence variation associated with antigenreceptors are found in the CDRs, these regions are sometimes referred toas hypervariable domains.

CDRs are found within loop regions of an antigen receptor (usuallybetween regions of β-sheet structure; see FIGS. 3A-3C). These loopregions are typically referred to as hypervariable loops. Each antigenreceptor comprises six hypervariable loops: H1, H2, H3, L1, L2 and L3.For example, the H1 loop comprises CDR1 of the heavy chain and the L3loop comprises CDR3 of the light chain. The CH2 and CH3 domain moleculesdescribed herein comprise engrafted amino acids from a variable domainof an antibody. The engrafted amino acids comprise at least a portion ofa CDR. The engrafted amino acids can also include additional amino acidsequence, such as a complete hypervariable loop. As used herein, a“functional fragment” of a CDR is at least a portion of a CDR thatretains the capacity to bind a specific antigen.

A numbering convention for the location of CDRs is described by Kabat etal., (1991) Sequences of Proteins of Immunological Interest, 5^(th)Edition, U.S. Department of Health and Human Services, Public HealthService, National Institutes of Health, Bethesda, Md. (NIH PublicationNo. 91-3242).

Contacting: Placement in direct physical association, which includesboth in solid and in liquid form.

Degenerate variant: As used herein, a “degenerate variant” of a CH2 orCH3 domain molecule is a polynucleotide encoding a CH2 or CH3 domainmolecule that includes a sequence that is degenerate as a result of thegenetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included as long as the amino acid sequence of the CH2 orCH3 domain molecule encoded by the nucleotide sequence is unchanged.

Domain: A protein structure which retains its tertiary structureindependently of the remainder of the protein. In some cases, domainshave discrete functional properties and can be added, removed ortransferred to another protein without a loss of function.

Effector molecule: A molecule, or the portion of a chimeric molecule,that is intended to have a desired effect on a cell to which themolecule or chimeric molecule is targeted. Effector molecule is alsoknown as an effector moiety (EM), therapeutic agent, or diagnosticagent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as a CH2 or CH3 domain molecule, may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (such as anantisense nucleic acid), or another therapeutic moiety that can beshielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art. See, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985.

Diagnostic agents or moieties include radioisotopes and other detectablelabels. Detectable labels useful for such purposes are also well knownin the art, and include radioactive isotopes such as ³²P, ¹²⁵I, and¹³¹I, fluorophores, chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groupsor contiguous or non-contiguous peptide sequences on a molecule that areantigenic, that is, that elicit a specific immune response. An antibodybinds a particular antigenic epitope based on the three dimensionalstructure of the antibody and the matching (or cognate) epitope.

Expression: The translation of a nucleic acid into a protein. Proteinsmay be expressed and remain intracellular, become a component of thecell surface membrane, or be secreted into the extracellular matrix ormedium

Expression control sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is an array of nucleic acid control sequences that directstranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. Both constitutive and inducible promoters are included(see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).

Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asthe metallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that containsa promoter sequence which facilitates the efficient transcription of theinserted genetic sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specificnucleic acid sequences that allow phenotypic selection of thetransformed cells.

Framework region: Amino acid sequences interposed between CDRs (orhypervariable regions). Framework regions include variable light andvariable heavy framework regions. Each variable domain comprises fourframework regions, often referred to as FR1, FR2, FR3 and FR4. Theframework regions serve to hold the CDRs in an appropriate orientationfor antigen binding. Framework regions typically form β-sheetstructures.

Fungal-associated antigen (FAAs): A fungal antigen which can stimulatefungal-specific T-cell-defined immune responses. Exemplary FAAs include,but are not limited to, an antigen from Candida albicans, Cryptococcus(such as d25, or the MP98 or MP88 mannoprotein from C. neoformans, or animmunological fragment thereof), Blastomyces (such as B. dermatitidis,for example WI-1 or an immunological fragment thereof), and Histoplasma(such as H. capsulatum).

Heterologous: A heterologous polypeptide or polynucleotide refers to apolypeptide or polynucleotide derived from a different source orspecies.

Hypervariable region: Regions of particularly high sequence variabilitywithin an antibody variable domain. The hypervariable regions form loopstructures between the β-sheets of the framework regions. Thus,hypervariable regions are also referred to as “hypervariable loops.”Each variable domain comprises three hypervariable regions, oftenreferred to as H1, H2 and H3 in the heavy chain, and L1, L2 and L3 inthe light chain. The loop structures of the hypervariable loops aredepicted in FIGS. 3A-5C.

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigen. An immune response can include any cell of the bodyinvolved in a host defense response for example, an epithelial cell thatsecretes an interferon or a cytokine. An immune response includes, butis not limited to, an innate immune response or inflammation.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody or a CH2 or CH3 domain molecule. The effector molecule can be adetectable label or an immunotoxin. Specific, non-limiting examples oftoxins include, but are not limited to, abrin, ricin, Pseudomonasexotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin(DT), botulinum toxin, or modified toxins thereof, or other toxic agentsthat directly or indirectly inhibit cell growth or kill cells. Forexample, PE and DT are highly toxic compounds that typically bring aboutdeath through liver toxicity. PE and DT, however, can be modified into aform for use as an immunotoxin by removing the native targetingcomponent of the toxin (such as domain Ia of PE and the B chain of DT)and replacing it with a different targeting moiety, such as a CH2 or CH3domain molecule. In one embodiment, a CH2 or CH3 domain molecule isjoined to an effector molecule (EM). In another embodiment, a CH2 or CH3domain molecule joined to an effector molecule is further joined to alipid or other molecule to a protein or peptide to increase itshalf-life in the body. The linkage can be either by chemical orrecombinant means. “Chemical means” refers to a reaction between the CH2or CH3 domain molecule and the effector molecule such that there is acovalent bond formed between the two molecules to form one molecule. Apeptide linker (short peptide sequence) can optionally be includedbetween the CH2 or CH3 domain molecule and the effector molecule.Because immunoconjugates were originally prepared from two moleculeswith separate functionalities, such as an antibody and an effectormolecule, they are also sometimes referred to as “chimeric molecules.”The term “chimeric molecule,” as used herein, therefore refers to atargeting moiety, such as a ligand, antibody or CH2 or CH3 domainmolecule, conjugated (coupled) to an effector molecule.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionucleotide or other molecule to apolypeptide, such as a CH2 or CH3 domain molecule. In the specificcontext, the terms include reference to joining a ligand, such as anantibody moiety, to an effector molecule (“EM”).

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating an immune response, such as theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule or protein) has been substantially separated or purified awayfrom other biological components from which the component naturallyoccurs (for example, other biological components of a cell), such asother chromosomal and extra-chromosomal DNA and RNA and proteins,including other antibodies. Nucleic acids and proteins that have been“isolated” include nucleic acids and proteins purified by standardpurification methods. An “isolated antibody” is an antibody that hasbeen substantially separated or purified away from other proteins orbiological components such that its antigen specificity is maintained.The term also embraces nucleic acids and proteins (including CH2 and CH3domain molecules) prepared by recombinant expression in a host cell, aswell as chemically synthesized nucleic acids or proteins, or fragmentsthereof.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or CH2 or CH3domain molecule, to facilitate detection of that molecule. Specific,non-limiting examples of labels include fluorescent tags, enzymaticlinkages, and radioactive isotopes.

Ligand contact residue or Specificity Determining Residue (SDR): Aresidue within a CDR that is involved in contact with a ligand orantigen. A ligand contact residue is also known as a specificitydetermining residue (SDR). A non-ligand contact residue is a residue ina CDR that does not contact a ligand. A non-ligand contact residue canalso be a framework residue.

Nanoantibody (nAb): A CH2 or CH3 domain molecule engineered such thatthe molecule specifically binds antigen. The CH2 and CH3 domainmolecules engineered to bind antigen are the smallest knownantigen-specific binding antibody domain-based molecules.

Neoplasia and Tumor: The product of neoplasia is a neoplasm (a tumor),which is an abnormal growth of tissue that results from excessive celldivision. Neoplasias are also referred to as “cancer.” A tumor that doesnot metastasize is referred to as “benign.” A tumor that invades thesurrounding tissue and/or can metastasize is referred to as “malignant.”

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma,astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma and retinoblastoma).

Examples of hematological tumors include leukemias, including acuteleukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2′-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together and can be made byartificially combining two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques.Recombinant nucleic acids include nucleic acid vectors comprising anamplified or assembled nucleic acid which can be used to transform asuitable host cell. A host cell that comprises the recombinant nucleicacid is referred to as a “recombinant host cell.” The gene is thenexpressed in the recombinant host cell to produce a “recombinantpolypeptide.” A recombinant nucleic acid can also serve a non-codingfunction (for example, promoter, origin of replication, ribosome-bindingsite and the like).

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pathogen: A biological agent that causes disease or illness to its host.Pathogens include, for example, bacteria, viruses, fungi, protozoa andparasites. Pathogens are also referred to as infectious agents.

Examples of pathogenic viruses include those in the following virusfamilies Retroviridae (for example, human immunodeficiency virus (HIV);human T-cell leukemia viruses (HTLV); Picornaviridae (for example, poliovirus, hepatitis A virus; hepatitis C virus; enteroviruses, humancoxsackie viruses, rhinoviruses, echoviruses; foot-and-mouth diseasevirus); Calciviridae (such as strains that cause gastroenteritis);Togaviridae (for example, equine encephalitis viruses, rubella viruses);Flaviridae (for example, dengue viruses; yellow fever viruses; West Nilevirus; St. Louis encephalitis virus; Japanese encephalitis virus; andother encephalitis viruses); Coronaviridae (for example, coronaviruses;severe acute respiratory syndrome (SARS) virus; Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, Ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus (RSV));Orthomyxoviridae (for example, influenza viruses); Bunyaviridae (forexample, Hantaan viruses; Sin Nombre virus, Rift Valley fever virus;bunya viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses; Machupo virus; Junin virus); Reoviridae(e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses; BK-virus);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV)-1 and HSV-2; cytomegalovirus (CMV); Epstein-Barr virus (EBV);varicella zoster virus (VZV); and other herpes viruses, includingHSV-6); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (such as African swine fever virus); Filoviridae (forexample, Ebola virus; Marburg virus); Caliciviridae (for example,Norwalk viruses) and unclassified viruses (for example, the etiologicalagents of Spongiform encephalopathies, the agent of delta hepatitis(thought to be a defective satellite of hepatitis B virus); andastroviruses).

Examples of fungal pathogens include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Examples of bacterial pathogens include, but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (such as. M. tuberculosis, M. avium, M. intracellulare,M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus anthracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, and Actinomyces israelli.

Other pathogens (such as protists) include: Plasmodium falciparum andToxoplasma gondii.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th

Edition (1975), describes compositions and formulations suitable forpharmaceutical delivery of one or more therapeutic compounds ormolecules, such as one or more antibodies, and additional pharmaceuticalagents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

The term “residue” or “amino acid residue” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.

“Conservative” amino acid substitutions are those substitutions that donot substantially affect or decrease an activity or antigenicity of apolypeptide. For example, a polypeptide can include at most about 1, atmost about 2, at most about 5, at most about 10, or at most about 15conservative substitutions and specifically bind an antibody that bindsthe original polypeptide. The term conservative variation also includesthe use of a substituted amino acid in place of an unsubstituted parentamino acid, provided that antibodies raised antibodies raised to thesubstituted polypeptide also immunoreact with the unsubstitutedpolypeptide. Examples of conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, and/or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity ofsigns or symptoms of a disease.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, and can be DNA oligonucleotides 15 nucleotides or more in length.Primers may be annealed to a complementary target DNA strand by nucleicacid hybridization to form a hybrid between the primer and the targetDNA strand, and then extended along the target DNA strand by a DNApolymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example, by the polymerase chain reaction(PCR) or other nucleic-acid amplification methods known in the art. Oneof skill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primercomprising 20 consecutive nucleotides will anneal to a target with ahigher specificity than a corresponding primer of only 15 nucleotides.Thus, in order to obtain greater specificity, probes and primers may beselected that comprise 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified CH2 or CH3domain molecule is one that is isolated in whole or in part fromnaturally associated proteins and other contaminants in which themolecule is purified to a measurable degree relative to its naturallyoccurring state, for example, relative to its purity within a cellextract or biological fluid.

The term “purified” includes such desired products as analogs ormimetics or other biologically active compounds wherein additionalcompounds or moieties are bound to the CH2 or CH3 domain molecule inorder to allow for the attachment of other compounds and/or provide forformulations useful in therapeutic treatment or diagnostic procedures.

Generally, substantially purified CH2 or CH3 domain molecules includemore than 80% of all macromolecular species present in a preparationprior to admixture or formulation of the respective compound withadditional ingredients in a complete pharmaceutical formulation fortherapeutic administration. Additional ingredients can include apharmaceutical carrier, excipient, buffer, absorption enhancing agent,stabilizer, preservative, adjuvant or other like co-ingredients. Moretypically, the CH2 or CH3 domain molecule is purified to representgreater than 90%, often greater than 95% of all macromolecular speciespresent in a purified preparation prior to admixture with otherformulation ingredients. In other cases, the purified preparation may beessentially homogeneous, wherein other macromolecular species are lessthan 1%.

Recombinant: A recombinant nucleic acid or polypeptide is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, for example, by genetic engineeringtechniques.

Sample: A portion, piece, or segment that is representative of a whole.This term encompasses any material, including for instance samplesobtained from a subject.

A “biological sample” is a sample obtained from a subject including, butnot limited to, cells, tissues and bodily fluids. Bodily fluids include,for example, saliva, sputum, spinal fluid, urine, blood and derivativesand fractions of blood, including serum and lymphocytes (such as Bcells, T cells and subfractions thereof). Tissues include those frombiopsies, autopsies and pathology specimens, as well as biopsied orsurgically removed tissue, including tissues that are, for example,unfixed, frozen, fixed in formalin and/or embedded in paraffin.

In particular embodiments, the biological sample is obtained from asubject, such as blood or serum. A biological sample is typicallyobtained from a mammal, such as a rat, mouse, cow, dog, guinea pig,rabbit, or primate. In one embodiment, the primate is macaque,chimpanzee, or a human.

Scaffold: As used herein, a CH2 or CH3 domain scaffold is a recombinantCH2 or CH3 domain that can be used as a platform to introduce mutations(such as into the loop regions; see FIG. 2 and FIGS. 3A-3C) in order toconfer antigen binding to the CH2 or CH3 domain. In some embodiments,the scaffold is altered to exhibit increased stability compared with thenative CH2 or CH3 domain. In particular examples, the scaffold ismutated to introduce pairs of cysteine residues to allow formation ofone or more non-native disulfide bonds. In some cases, the scaffold is aCH2 or CH3 domain having an N-terminal deletion, such as a deletion ofabout 1 to about 7 amino acids.

Sequence identity: The similarity between nucleotide or amino acidsequences is expressed in terms of the similarity between the sequences,otherwise referred to as sequence identity. Sequence identity isfrequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants will possess a relatively high degree ofsequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higginsand Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994.

The NCBI Basic Local Alignment Search Tool (BLAS™) (Altschul et al., J.Mol. Biol. 215:403-410, 1990.) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Specific binding agent: An agent that binds substantially only to adefined target. Thus an antigen specific binding agent is an agent thatbinds substantially to an antigenic polypeptide or antigenic fragmentthereof. In one embodiment, the specific binding agent is a monoclonalor polyclonal antibody or a CH2 or CH3 domain molecule that specificallybinds the antigenic polypeptide or antigenic fragment thereof.

The term “specifically binds” refers, with respect to an antigen, to thepreferential association of an antibody or other ligand, in whole orpart, with a cell or tissue bearing that antigen and not to cells ortissues lacking a detectable amount of that antigen. It is, of course,recognized that a certain degree of non-specific interaction may occurbetween a molecule and a non-target cell or tissue. Nevertheless,specific binding may be distinguished as mediated through specificrecognition of the antigen. Specific binding results in a much strongerassociation between the antibody (or CH2 or CH3 domain molecule) andcells bearing the antigen than between the bound antibody (or CH2 or CH3domain molecule) and cells lacking the antigen. Specific bindingtypically results in greater than 2-fold, such as greater than 5-fold,greater than 10-fold, or greater than 100-fold increase in amount ofbound antibody or CH2 or CH3 domain molecule (per unit time) to a cellor tissue bearing the antigenic polypeptide as compared to a cell ortissue lacking the antigenic polypeptide respectively. Specific bindingto a protein under such conditions requires an antibody or CH2 or CH3domain molecule that is selected for its specificity for a particularprotein. A variety of immunoassay formats are appropriate for selectingantibodies or CH2 or CH3 domain molecules specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Therapeutically effective amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. Such agents include the CH2 or CH3 domain moleculesdescribed herein. For example, this may be the amount of an H1V-specificCH2 domain molecule useful in preventing, treating or amelioratinginfection by HIV. Ideally, a therapeutically effective amount of anantibody is an amount sufficient to prevent, treat or ameliorateinfection or disease, such as is caused by HIV infection in a subjectwithout causing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount of an agent useful for preventing,ameliorating, and/or treating a subject will be dependent on the subjectbeing treated, the type and severity of the affliction, and the mannerof administration of the therapeutic composition.

Toxin: A molecule that is cytotoxic for a cell. Toxins include, but arenot limited to, abrin, ricin, Pseudomonas exotoxin (PE), diphtheriatoxin (DT), botulinum toxin, saporin, restrictocin or gelonin, ormodified toxins thereof. For example, PE and DT are highly toxiccompounds that typically bring about death through liver toxicity. PEand DT, however, can be modified into a form for use as an immunotoxinby removing the native targeting component of the toxin (for example,domain Ia of PE or the B chain of DT) and replacing it with a differenttargeting moiety, such as a CH2 or CH3 domain molecule.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Tumor-associated antigens (TAAs): A tumor antigen which can stimulatetumor-specific T-cell-defined immune responses. Exemplary TAAs include,but are not limited to, RAGE-1, tyrosinase, MAGE-1, MAGE-2, NY-ESO-1,Melan-A/MART-1, glycoprotein (gp) 75, gp100, beta-catenin, PRAME, MUM-1,WT-1, CEA, and PR-1. Additional TAAs are known in the art (for examplesee Novellino et al., Cancer Immunol. Immunother. 54(3):187-207, 2005)and includes TAAs not yet identified.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Viral-associated antigen (VAAs): A viral antigen which can stimulateviral-specific T-cell-defined immune responses. Exemplary VAAs include,but are not limited to, an antigen from human immunodeficiency virus(HIV), BK virus, JC virus, Epstein-Ban virus (EBV), cytomegalovirus(CMV), adenovirus, respiratory syncytial virus (RSV), herpes simplexvirus 6 (HSV-6), parainfluenza 3, or influenza B.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

Conventional antibodies are large multi-subunit protein complexescomprising at least four polypeptide chains, including two light chainsand two heavy chains (see FIG. 1A for a schematic drawing of aconventional immunoglobulin molecule). The heavy and light chains ofantibodies contain variable regions, which bind antigen, and constantregions (such as CH1, CH2 and CH3 domains), which provide structuralsupport and effector functions. The antigen binding region comprises twoseparate domains, a heavy chain variable domain (V_(H)) and a lightchain variable domain (V_(L)). A typical antibody, such as an IgGmolecule, has a molecular weight of approximately 150 kD. A number ofsmaller antigen binding fragments of naturally occurring antibodies havebeen identified following protease digestion (for example, Fab, Fab′,and F(ab′)₂). These antibody fragments have a molecular weight rangingfrom approximately 50 to 100 kD. Recombinant methods have been used togenerate alternative antigen-binding fragments, termed single chainvariable fragments (scFv), which consist of V_(L) and V_(H) joined by asynthetic peptide linker. A scFv molecule has a molecular weight ofapproximately 25-30 kD.

However, in some cases, therapeutic use of antibodies or antibodyfragments can be limited due to the size of the antibody. For example,if an antibody or antibody fragment is too large, tissue penetration andepitope access may be restricted. In addition, many therapeuticantibodies are of non-human origin, which can result in toxicity in ahuman subject. Given these limitations, small, human antibodies that canspecifically bind antigen are desirable for diagnostic or therapeuticapplications that utilize antibodies or their fragments.

Described herein are engineered antibody constant domain molecules.Disclosed herein are recombinant CH2 and CH3 domain molecules that serveas scaffolds for the introduction of mutations to confer antigen bindingto the molecule. Also provided are the modified CH2 and CH3 domainmolecules that specifically bind antigen. In some embodiments, theantibody constant domain is a CH2 domain from IgG, IgA or IgD. In otherembodiments, the antibody constant domain is a CH3 domain from IgE orIgM. The disclosed CH2 and CH3 domain molecules are small, stable,soluble, have minimal to no toxicity and in some cases, are capable ofbinding antigen. The CH2 and CH3 domain molecules described herein donot comprise more than one constant domain and do not compriseimmunoglobulin variable domains.

Provided herein are polypeptides comprising an immunoglobulin CH2 or CH3domain, wherein the CH2 or CH3 domain comprises at least onecomplementarity determining region (CDR), or a functional fragmentthereof (such as a SDR), from a heterologous immunoglobulin variabledomain. Also provided are CH2 or CH3 domain molecules comprising atleast one mutation, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremutations in one or more loops of the CH2 or CH3 domain. The CH2 or CH3domain molecules described herein have a molecular weight of less thanabout 15 kD. In some embodiments, the CH2 or CH3 domain molecules have amolecular weight of about 12 to about 14 kD. In some embodiments, theCH2 or CH3 domains comprise an N-terminal truncation of about 1 to about7 amino acids, such as 1, 2, 3, 4, 5, 6 or 7 amino acids. In someembodiments, the CH2 or CH3 domain molecules comprise a C-terminaltruncation of about 1 to about 4 amino acids, such as 1, 2, 3 or 4 aminoacids.

Introduction of specific mutations and/or engraftment of theheterologous CDR to the CH2 or CH3 domain enables the polypeptide tobind antigen. In some embodiments, the engrafted portion from theheterologous immunoglobulin comprises only a CDR, or functional fragmentthereof. In other embodiments, the engrafted portion comprisesadditional sequence, such as all or a portion of the hypervariable loop.The length of the engrafted portion can vary, but is typically between 5and 21 amino acids, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or 21 amino acids. In one embodiment, the engraftedportion is between 8 and 15 amino acids. Although the length of theengrafted portion varies, the resulting CH2 or CH3 domain moleculespecifically binds antigen. In some embodiments, the CH2 or CH3 domainmolecules specifically binds an antigen with a K_(d) of about 10⁻⁶,about 10⁻⁷ or about 10⁻⁸ M. In some embodiments, the polypeptidecomprises more than one CDR, or functional fragment thereof, such as twoor three CDRs.

In some embodiments, at least a portion of a loop region of the CH2 orCH3 domain is replaced by the CDR or functional fragment thereof. Thenumber of amino acids removed from the loop region can vary. In someembodiments, the number of amino acids removed from the loop region isbetween 1 and 10 amino acids, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids. In other embodiments, the CDR is engrafted without removingamino acids from the loop region. The number of amino acids removed fromthe CH2 or CH3 domain loop or loops can vary. One of skill in the art iscapable of determining the appropriate sequence to remove empirically,such as by testing the CH2 or CH3 domain molecules for stability,solubility and the capacity to bind an antigen of interest.

The particular CDR engrafted can be any CDR from any immunoglobulinvariable domain, such as a V_(H) domain or a V_(L) domain. In oneembodiment, the CDR is CDR1. In another embodiment, the CDR is CDR2. Inanother embodiment, the CDR is CDR3. In other embodiments, two or threeor more CDRs are engrafted in the loops of the CH2 or CH3 domainmolecule.

The CH2 or CH3 domain loop replaced by the CDR (or the CH2 domain intowhich the CDR is engrafted, without removal of loop sequence) can be anyloop of the CH2 or CH3 domain. In one embodiment, the loop region isselected from Loop 1, Loop 2, Loop 3, Loop A-B, Loop C-D or Loop E-F.Any loop of the CH2 or CH3 domain can be replaced by any CDR. Inaddition, multiple loops can be replaced by CDRS, in any combination. Inone embodiment, Loop 1 is replaced by CDR1 or CDR3. In anotherembodiment, Loop 3 is replaced by CDR1 or CDR3. In another embodiment,Loop 1 and Loop 3 are replaced by CDR1 and CDR3, respectively. Inanother embodiment, Loop 1 and Loop 3 are replaced by CDR3 and CDR1,respectively. In other embodiments, Loop A-B is replaced by CDR1; LoopC-D is replaced by CDR2; or Loop E-F is replaced by CDR3.

In preferred embodiments, the polypeptides provided herein do notcomprise a variable domain, such as a V_(H) domain or a V_(L) domain.

The antibody constant domain can be derived from any type ofimmunoglobulin. In one embodiment, the immunoglobulin is an IgG. Inother embodiments, the immunoglobulin is an IgA, IgD, IgM or IgE. Inparticular examples, the constant domain is a CH2 domain from IgG.

In some embodiments, the CH2 or CH3 domains that bind antigen haveadditional mutations that increase stability of the molecule. Forexample, the molecules can comprise mutations that allow for theformation of non-native disulfide bonds, such as by introducing a pairof amino acid substitutions to replace original residues with cysteineresidues. In some examples, a first amino acid substitution isintroduced in the N-terminal A strand and the second amino acidsubstitution is introduced in the C-terminal G strand of the constantdomain. In addition, the antigen binding CH2 and CH3 domain moleculescan be either glycosylated or unglycosylated.

Also provided herein are polypeptides comprising an immunoglobulin CH2domain of IgG, Ig or IgD, or a CH3 domain of IgE and IgM, wherein theCH2 domain or CH3 domain comprises a first amino acid substitution and asecond amino acid substitution, wherein the first and second amino acidsubstitutions each replace the original residue with a cysteine residue,wherein the cysteine residues form a disulfide bond, and wherein thepolypeptide has a molecular weight of less than about 15 kD. Such CH2and CH3 domains exhibit increased stability relative to unmodified CH2and CH3 domains, and thus are useful as scaffolds for introducingmutations to confer antigen binding to the CH2 or CH3 domain.

In some embodiments, the first amino acid substitution is in theN-terminal A strand and the second amino acid substitution is in theC-terminal G strand, which allows formation of a disulfide bond betweenthe A and G strands (see FIGS. 3A-3C for a schematic of the loopregions). In some examples, the constant domain is a CH2 domain of IgG.

In particular examples described herein, the first amino acidsubstitution is L12 to C12 and the second amino acid substitution isK104 to C104 (numbered with reference to SEQ ID NO: 5). In otherexamples, the first amino acid substitution is V10 to C10 and the secondamino acid substitution is K104 to C104 (numbered with reference to SEQID NO: 5).

The CH2 and CH3 domain scaffold can comprise additional mutations, suchas to increase stability or enhance solubility and expression. In someembodiments, the CH2 or CH3 domain comprises an N-terminal truncation ofabout 1 to about 7 amino acids. In particular examples, the N-terminaltruncation is 1, 2, 3, 4, 5, 6 or 7 amino acids in length. In someembodiments, the CH2 or CH3 domain scaffold comprises a C-terminaltruncation of about 1 to about 4 amino acids. In particular examples,the C-terminal truncation is 1, 2, 3 or 4 amino acids.

In some embodiments, the CH2 or CH3 domain scaffold is further mutatedto confer antigen binding. In particular embodiments, (i) at least oneof the loops of the CH2 or CH3 domain is mutated; (ii) at least aportion of a loop region of the CH2 or CH3 domain is replaced by a CDRor fragment thereof from a heterologous immunoglobulin variable domain;or (iii) both.

In addition, the CH2 domain or CH3 domain can either be unglycosylatedor glycosylated. For example, a recombinant CH2 or CH3 domain can beexpressed in a mammalian cell to allow for post-translationalmodifications, such as glycosylation.

In some embodiments, the antigen is from a pathogen, such as a virus orbacterium. In one embodiment, the pathogen is HIV. In other embodiments,the antigen is a cancer-specific antigen or a cancer-related protein. Inother embodiments, the antigen is related to an autoimmune disease (forexample, TNF-α).

In some embodiments, the CH2 or CH3 domain molecule binds a tumorantigen. The tumor antigen can be any tumor-associated antigen, whichare well known in the art.

Also provided herein are compositions comprising the CH2 or CH3 domainmolecules described herein. In some embodiments, the compositioncomprises a CH2 domain or CH3 domain and a pharmaceutically acceptablecarrier.

Nucleic acid molecules encoding the disclosed CH2 or CH3 domainmolecules, vectors comprising the nucleic acid sequences, and cellscomprising the vectors are also provided herein.

In some embodiments, engineered CH2 or CH3 domain molecules comprise Fcreceptor binding sites and are capable of binding at least one Fcreceptor. In particular examples, the Fc receptor is the neonatal Fcreceptor. The ability to bind an Fc receptor confers effector functionsto the CH2 or CH3 domain molecule, such as, for example, ADCC. In otherembodiments, engineered CH2 or CH3 domains bind complement-relatedmolecules, such as C1q, which can activate the compliment system. In yetother embodiments, the CH2 or CH3 domain molecules are conjugated to aneffector molecule, which include, but are not limited to, therapeutic,diagnostic, or detection moieties.

Further provided are methods of use of the CH2 or CH3 domain moleculesfor the preparation of a medicament. In one embodiment, the medicamentis for the treatment of HIV infection. In another embodiment, themedicament is for the treatment of cancer. In another embodiment, themedicament is for the treatment of an autoimmune or inflammatorydisorder.

The CH2 and CH3 domain molecules described herein can be engineered tospecifically bind any desired antigen. Methods of identifying andselecting antigen-specific CH2 or CH3 domain molecules can be achievedusing any suitable technique known in the art, such as by using a phagedisplay library.

Provided herein is a method of identifying a recombinant CH2 domain orCH3 domain that specifically binds a target antigen. The method includes(a) providing a library of particles displaying on their surface arecombinant CH2 or CH3 domain, wherein the CH2 or CH3 domain has amolecular weight less than about 15 kD; (b) contacting the library ofparticles with the target antigen to select particles that specificallybind the target antigen; and (c) cloning the CH2 or CH3 domain nucleicacid molecules from the particles expressing the CH2 or CH3 domains thatspecifically bind the target antigen, thereby identifying a CH2 or CH3domain that specifically binds the target antigen. In some embodiments,the library is generated by (i) providing a library of nucleic acidmolecules encoding a genetically diverse population of CH2 or CH3domains, wherein the genetically diverse population is provided byintroducing mutations into one or more loop regions of the CH2 or CH3domain; and (ii) expressing the library of nucleic acid molecules inrecombinant host cells, whereby the CH2 domains or CH3 domains areexpressed on the surface of the particles and the CH2 or CH3 domainnucleic acid molecules are encoded by the genetic material of theparticles. In some embodiments, the CH2 or CH3 domain comprises anN-terminal deletion of about 1 to about 7 amino acids. In someembodiments, the particles are phage particles.

In some embodiments, the phage library expresses recombinant CH2domains, such as IgG CH2 domains. In some embodiments, the CH2 domain orCH3 domains comprise at least one mutation in Loop 1, or at least onemutation in Loop 2, or at least one mutation in Loop 3, or at least onemutation in Loop A-b, or at least one mutation in Loop C-D, or at leastone mutation in Loop E-F, or any combination thereof.

Any suitable recombinant host cell can be used to generate phageparticles. Such host cells are well known in the art. In some examples,the recombinant host cells are TG1 cells.

Further provided herein is a method of making a library of recombinantCH2 or CH3 domains, comprising (i) introducing mutations into one ormore loop regions of a CH2 domain or CH3 domain scaffold, or (ii)replacing a portion of a loop region of the CH2 domain or CH3 domainscaffold with a CDR or functional fragment thereof from a heterologousimmunoglobulin variable domain, or (iii) both, wherein the scaffoldcomprises an isolated immunoglobulin CH2 domain of IgG, IgA or IgD orCH3 domain of IgE or IgM.

In some embodiments, the CH2 or CH3 domain scaffold further comprises anN-terminal truncation of about 1 to about 7 amino acids, such as about1, 2, 3, 4, 5, 6 or 7 amino acids. In some embodiments, the CH2 or CH3domain scaffold further comprises a C-terminal truncation of about 1 toabout 4 amino acids, such as about 1, 2, 3 or 4 amino acids.

In some cases, the CH2 or CH3 domain scaffold further comprisesadditional mutations to stabilize the molecule. In some embodiments ofthe method, the CH2 or CH3 domain scaffold further comprises a firstamino acid substitution and a second amino acid substitution, whereinthe first and second amino acid substitutions each replace the originalresidue with a cysteine residue, wherein the cysteine residues form adisulfide bond.

Further provided is a method of identifying a recombinant CH2 domain orCH3 domain that specifically binds a target antigen, comprisingcontacting the library produced by the methods disclosed herein with thetarget antigen to select recombinant CH2 or CH3 domains thatspecifically bind the target antigen.

Also provided are libraries, such as phage-displayed libraries, of CH2or CH3 domain molecules. The libraries comprise CH2 or CH3 domainmolecules having one or more mutations, engrafted CDRs, hypervariableloops, or functional fragments thereof. The libraries comprising mutatedresidues can be used to identify CH2 or CH3 domain molecules having adesired antigen binding affinity and/or to identify CH2 or CH3 domainmolecules with reduced immunogenicity.

Further provided are kits comprising the CH2 or CH3 domain moleculesdisclosed herein. In one embodiment, the CH2 or CH3 domain molecule islabeled (such as with a fluorescent, radioactive, or an enzymaticlabel). In another embodiment, a kit includes instructional materialsdisclosing means of use of a CH2 or CH3 domain molecule. Theinstructional materials may be written, in an electronic form (forexample computer diskette or compact disk) or may be visual (such asvideo files). The kits can also include additional components tofacilitate the particular application for which the kit is designed.Thus, for example, the kit can additionally contain means of detecting alabel (such as enzyme substrates for enzymatic labels, filter sets todetect fluorescent labels, appropriate secondary labels such as asecondary antibody, or the like). The kits can additionally includebuffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

IV. Engineered Antibody Constant Domains

The engineered antibody constant domain molecules described herein aresmall in size (typically less than 15 kD), which offers significantadvantages for detection, diagnosis and treatment. For example, thesmall size of the molecules allows for greater epitope access and bettertissue penetration. As shown in FIG. 5C, the CH2 domain antibodiesprovided herein have a lower molecular weight than other types ofantibodies and antibody fragments, such as scFv, Fab and IgG molecules.They are also smaller than V_(H) domain antibodies.

As described herein, the CH2 or CH3 domain molecules can effectivelybind antigen in the absence of other immunoglobulin domains, includingvariable domains or other constant domains. For example, the CH2 or CH3domain molecules can specifically bind an antigen with a kD of about10⁻⁶, about 10⁻⁷, about 10⁻⁸ or about 10⁻⁹ or less.

The CH2 or CH3 domains described herein that specifically bind anantigen comprise at least one heterologous amino acid sequence from animmunoglobulin variable domain, and/or comprise at least one mutation.The heterologous amino acid sequence engrafted in the CH2 or CH3 domaincomprises at least one CDR, or functional fragment thereof (such as anSDR from an antibody that specifically binds an antigen of interest).The engrafted amino acid sequence can also contain additional amino acidsequence extending from the CDR toward the N-terminus and/or toward theC-terminus, such as other amino acids comprising the hypervariable loop.Thus, in some embodiments, the engineered CH2 or CH3 domain moleculescomprise a complete hypervariable loop from a heterologousimmunoglobulin variable domain.

The engineered CH2 and CH3 domains can further comprise second or thirdCDRs or hypervariable loops. The length of the engrafted CDR orhypervariable loop can vary. Appropriate lengths can be determinedempirically, such as by expressing the engineered CH2 or CH3 domains andassessing stability and solubility of the protein, as well as bydetermining binding affinity. Methods of protein expression, determiningprotein solubility and evaluating antigen binding affinity are wellknown in the art. As described herein, it has been determined thatsequences up to 21 amino acids in length can be successfully engraftedin the CH2 domain.

A human CH2 domain comprises six loop regions: Loop 1, Loop 2, Loop 3,Loop A-B, Loop C-D and Loop E-F. CDRs and/or hypervariable loops from aheterologous immunoglobulin variable domain can be engrafted in one ormore of any of these loops, in any combination (see FIGS. 5A-5C forexamples).

The amino acid sequence of the human γ1 CH2 domain is set forth as SEQID NO: 5. The amino acid residues comprising each of the loop regions isshown below in Table 1. The amino acid positions are numbered startingwith number 1 for the first residue of the CH2.

TABLE 1 Amino Acid Positions of CH2 Domain Loops Amino acid positionsLoop (SEQ ID NO: 5) Loop A-B 14-27 Loop 1 35-43 Loop C-D 54-62 Loop 267-69 Loop E-F 78-88 Loop 3  96-100

The amino acid sequence of the human V_(H) domain is shown in FIG. 1B,and set forth as SEQ ID NO: 1. The amino acid residues comprising eachCDR and hypervariable loop is shown below in Table 2.

TABLE 2 Amino Acid Positions of Hypervariable Loops Amino acid positionsCDR/Loop (SEQ ID NO: 1) H1/CDR1 27-36 H2/CDR2 50-68 H3/CDR3  99-109

In one exemplary embodiment, nine amino acids from Loop 1 of the CH2domain are replaced with 10 amino acids from hypervariable loop H1/CDR1from the V_(H) domain of a human antibody. In other exemplaryembodiments, six amino acids from Loop 3 of the CH2 domain are replacedwith twelve or thirteen amino acids from hypervariable loop H3/CDR3 ofthe V_(H) domain of a human antibody. In another exemplary embodiment,six amino acids from Loop 3 of the CH2 domain are replaced with 10 aminoacids from hypervariable loop H1/CDR1 from the V_(H) domain of a humanantibody. In other exemplary embodiments, nine amino acids from Loop 1of the CH2 domain are replaced with twelve or thirteen amino acids fromhypervariable loop H3/CDR3 of the V_(H) domain of a human antibody.

In other embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids ofone or more of Loops 1, 2, 3, A-B, C-D or E-F are replaced with 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids ofone or more CDRs or hypervariable loops from a heterologous antibody, inany combination. The CDR or hypervariable loops can be from a V_(L) or aV_(H) domain (see FIGS. 3A-3C).

The engrafted hypervariable loop(s) or CDR(s) can be from any antibodyof interest. Such antibodies include, but are not limited to,pathogen-specific antibodies and cancer-specific antibodies.Pathogen-specific antibodies, include for example, antibodies thatspecifically bind an antigen from a pathogen such as viruses, bacteriaor fungi, protozoa or parasites. In one exemplary embodiment, theantibody specifically binds HIV-1. Cancer-specific antibodies includeantibodies that specifically recognize antigen expressed (such as on thecell surface) by the cancer cell, but not by other non-cancer cells.Examples of cancer-specific antibodies, include, but are not limited to,antibodies that recognize lung cancer, breast cancer, prostate cancer,liver cancer, bladder cancer, thyroid cancer, kidney cancer, pancreaticcancer, colorectal cancer, skin cancer, melanoma, neuroblastoma, Ewing'ssarcoma, leukemia or lymphoma cells or tissue.

In some embodiments, the engineered CH2 or CH3 domain molecules compriseCDR/hypervariable sequence with a known specificity. Alternatively, theengineered CH2 domain molecules can comprise randomized CDR peptidesequence or sequences. Mutational analysis of the CDRs can be performedto identify CH2 domain molecules having increased binding affinityand/or decreased immunogenicity. In addition, libraries of CH2 or CH3domain molecules comprising randomized or mutated CDR peptide sequencescan be generated to identify CH2 or CH3 domain molecules that bind withhigh affinity to a particular antigen of interest, such as describedbelow.

The CH2 and CH3 domain molecules provided herein can further compriseeffector molecules, such as for therapeutic, diagnostic or detectionpurposes. For example, effector molecules can include toxins anddetectable labels, such as radiolabels, enzymes or fluorescent markers.Additional details on the types of effector molecules that can be usedwith CH2 and CH3 domain molecules is described below (see “EffectorFunctions of Antibody Constant Domain Molecules”).

V. Antibody Constant Domain Molecule Libraries

Further provided herein are libraries of engineered CH2 or CH3 domainmolecules comprising randomly inserted or mutated CDR amino acidsequences. The libraries can be used to screen for CH2 or CH3 domainmolecules having high affinity for a particular antigen of interest. Inone embodiment, the libraries are phage display libraries. Antibodyphage display libraries, and methods of generating such libraries, arewell known in the art (see, for example, U.S. Pat. Nos. 6,828,422 and7,195,866, incorporated herein by reference).

The development of libraries of polypeptides, including antibodies, hasbeen described (U.S. Pat. No. 6,828,422). To generate a library ofpolypeptides (such as a library of CH2 or CH3 domain molecules), nucleicacid sequences suitable for the creation of the libraries must first begenerated. To generate such randomized nucleic acid sequences, typicallyerror-prone PCR is used. Mutations are introduced randomly in at leastone of the loops. For example, a collection (such as two or three ormore) of homologous proteins is identified, a database of the proteinsequences is established and the protein sequences are aligned to eachother. In the case of CH2 domain molecules, a collection of human CH2domain sequences are identified and used to create the database. Thedatabase is used to define subgroups of protein sequences whichdemonstrate a high degree of similarity in the sequence and/orstructural arrangement. For each of the subgroups, a polypeptidesequence comprising at least one consensus sequence is deduced whichrepresents the members of this subgroup (such as a subgroup of CH2domains). The complete collection of polypeptide sequences representsthe complete structural repertoire of the collection of homologousproteins (the CH2 domains). These artificial polypeptide sequences canbe analyzed according to their structural properties to identifyunfavorable interactions between amino acids within the polypeptidesequences or between the polypeptide sequences and other polypeptidesequences. Such interactions can be removed by changing the consensussequence accordingly.

Next, the polypeptide sequences are analyzed to identify sub-elements,including domains, loops, β-sheets, α-helices and/or CDRs. The aminoacid sequence is back translated into a corresponding coding nucleicacid sequence which is adapted to the codon usage of the host plannedfor expressing the described nucleic acid sequences. A set of cleavagesites is set up such that each of the sub-sequences encoding thesub-elements identified as described above, is flanked by two siteswhich do not occur a second time within the nucleic acid sequence. Thiscan be achieved by either identifying a cleavage site already flanking asub-sequence or by changing one or more nucleotides to create thecleavage site, and by removing that site from the remaining part of thegene. The cleavage sites should be common to all correspondingsub-elements or sub-sequences, which allows for the creation of a fullymodular arrangement of the sub-sequences in the nucleic acid sequenceand of the sub-elements in the corresponding polypeptide.

The nucleic acid sequences described above are synthesized using any oneof several methods well known in the art, such as, for example, by totalgene synthesis or by PCR-based approaches.

In one embodiment, the nucleic acid sequences are cloned into a vector.The vector can be a sequencing vector, an expression vector or a displayvector (such as a phage display), which are well known in the art.Vectors can comprise one nucleic acid sequence, or two or more nucleicsequences, either in a different or the same operon. If in the sameoperon, the nucleic acid sequences can be cloned separately or ascontiguous sequences.

In one embodiment, one or more sub-sequences (such as a loop) of thenucleic acid sequences are replaced by different sequences. This can beachieved by excising the sub-sequences using the cleavage sites adjacentto or at the end of the sub-sequence, such as by an appropriaterestriction enzyme, and replacing the sub-sequence by a differentsequence compatible with the cleaved nucleic acid sequence. In a furtherembodiment, the different sequences replacing the initialsub-sequence(s) (also referred to as “engrafted sequences”) are genomicor rearranged genomic sequences, for example CDRs, SDRs or hypervariableloops from a heterologous antibody. In some embodiments, theheterologous sequences are random sequences. The introduction of randomsequences introduces variability into the polypeptides (or CH2 domainmolecules) to create a library. The random sequences can be generatedusing any of a number of methods well known in the art, such as by usinga mixture of mono- or tri-nucleotides during automated oligonucleotidesynthesis or by error-prone PCR. The random sequences can be completelyrandomized or biased toward or against certain codons according to theamino acid distribution at certain positions in known protein sequences.Additionally, the collection of random sub-sequences can comprisedifferent numbers of codons, giving rise to a collection of sub-elementshaving different lengths.

The nucleic acid sequences can be expressed from a suitable vector underappropriate conditions well known in the art. In one embodiment, thepolypeptides expressed from the nucleic acid sequences are screened. Thepolypeptides can further be optimized. Screening can be performed byusing any method well known in the art, such as phage-display,selectively infective phage, polysome technology to screen for binding,assay systems for enzymatic activity or protein stability. Polypeptides(such as CH2 domain molecules) having the desired property can beidentified by sequencing the nucleic acid sequence or amino acidsequence, or by mass spectrometry. The desired property the polypeptidesare screened for can be, for example, optimized affinity or specificityfor a target molecule.

In some embodiments, phagemid vectors can be used to simultaneouslyexpress a large number of nucleic acid sequences, such as those encodinga library of CH2 or CH3 domain molecules (see, for example, U.S. PatentApplication Publication No. 2008/0312101). The libraries of phageparticles expressing CH2 and CH3 domains can be screened using anyscreening assay known to be applicable with phage. For example, thephage can be exposed to a purified antigen, soluble or immobilized (e.g.on a plate or on beads) or exposed to whole cells, tissues, or animals,in order to identify phage that adhere to targets present in complexstructures, and in particular in physiologically or therapeuticallyrelevant locations (e.g. binding to cancer cells or to an antigen on aviral particle).

The selected phagemid vectors in which a heterologous sequence has beencloned, expressed, and specifically isolated on the basis of its bindingfor a specific ligand, can be extracted from the bacterial cells, andsequenced, PCR-amplified, and/or recloned into another appropriatevector, for example for the large scale recombinant production inbacterial, plant, yeast, or mammalian cells.

The detection of the interaction with the specific target antigen can beperformed by applying standard panning methods, or by applying moresophisticated biophysical technologies for assessment of interactionsbetween the displayed CH2 or CH3 binding molecule and its targetantigen, such as fluorescence-based spectroscopy or microscopy,phosphatase reaction, or other high-throughput technologies.

Once CH2 or CH3 domain-expressing phage particles that specifically binda target antigen have been selected, the recombinant phage and therelevant DNA sequence can be isolated and characterized according to themethods known in the art (e.g. separated from the phagemid vector usingrestriction enzymes, directly sequenced, and/or amplified by PCR). Thesesequences can be then transferred into more appropriate vectors forfurther modification and/or expression into prokaryotic or eukaryotichost cells. The DNA sequence coding for the CH2 or CH3 domain, onceinserted into a suitable vector, can be introduced into appropriate hostcells by any suitable means (transformation, transfection, conjugation,protoplast fusion, electroporation, calcium phosphate precipitation,direct microinjection, etc.) to transform the cells.

This collection of DNA molecules can then be used to create libraries ofCH2 or CH3 domain molecules. The affinity of the CH2 or CH3 domainmolecules can be optimized using the methods described above. Thelibraries can be used to identify one or more CH2 or CH3 domainmolecules that bind to a target. Identification of the desired CH2 orCH3 domain molecules comprises expressing the CH2 or CH3 domainmolecules and then screening them to isolate one or more molecules thatbind to a given target molecule with the desired affinity. If necessary,the modular design of the DNA molecules allows for excision of one ormore genetic sub-sequences and replacement with one or more secondsub-sequences encoding structural sub-elements. The expression andscreening steps can then be repeated until a CH2 or CH3 domain moleculehaving the desired affinity is generated.

In one embodiment is a method in which one or more of the geneticsubunits (for example, one or more CH2 or CH3 domain loop regions) arereplaced by a random collection of sequences (the library) using thecleavage sites. The resulting library is then screened against anychosen antigen. CH2 or CH3 domain molecules with the desired properties(such as having the desired binding affinity) are selected, collectedand can be used as starting material for the next library.

In another embodiment, fusion proteins can be generated by providing aDNA sequence which encodes both the polypeptide, as described above, andan additional moiety. Such moieties include immunotoxins, enzymes,effector molecules, therapeutic molecules, labels or tags (such as fordetection and/or purification).

Also provided herein are the nucleic acid sequences, vectors containingthe nucleic acid sequences, host cells containing the vectors, andpolypeptides, generated according to the methods described above.

Further provided are kits comprising one or more of the nucleic acidsequences, recombinant vectors, polypeptides, and/or vectors accordingto the methods described above, and suitable host cells for producingthe polypeptides.

VI. Nucleic Acids Encoding Antibody Constant Domain Molecules

Nucleic acid sequences encoding the CH2 or CH3 domain molecules and/orimmunotoxins can be prepared by any suitable method including, forexample, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, using an automated synthesizer asdescribed in, for example, Needham-VanDevanter et al., Nucl. Acids Res.12:6159-6168, 1984; and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding a CH2 or CH3 domainmolecule, or an immunotoxin including a CH2 or CH3 domain molecule, canbe prepared by cloning techniques. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through many cloning exercises are found in Sambrook et al.,supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill

In one example, a CH2 domain molecule of use is prepared by insertingthe cDNA which encodes the CH2 domain molecule into a vector whichcomprises the cDNA encoding an effector molecule (EM). The insertion ismade so that the variable region and the EM are read in frame so thatone continuous polypeptide is produced. Thus, the encoded polypeptidecontains a functional CH2 domain region and a functional EM region. Inone embodiment, cDNA encoding an effector molecule, such as, but notlimited to a cytotoxin, is ligated to a CH2 domain molecule so that theEM is located at the carboxyl terminus of the CH2 domain molecule. Inone example, cDNA encoding a Pseudomonas exotoxin (“PE”), mutated toeliminate or to reduce non-specific binding, is ligated to a CH2 domainmolecule so that the EM is located at the amino terminus of the CH2domain molecule

Once the nucleic acids encoding the CH2 domain molecule (or immunotoxin)are isolated and cloned, the protein can be expressed in recombinantlyengineered cells such as bacteria, plant, yeast, insect or mammaliancells. For example, one or more DNA sequences encoding the CH2 domainmolecule can be expressed in vivo by DNA transfer into a suitable hostcell. The cell may be prokaryotic or eukaryotic. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart. Alternatively the DNA sequences encoding the immunotoxin, antibody,or fragment thereof can be expressed in vitro.

Polynucleotide sequences encoding the CH2 or CH3 domain molecules can beoperatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to appropriate promoters,enhancers, transcription terminators, a start codon (such as ATG) infront of a protein-encoding gene, splicing signal for introns,maintenance of the correct reading frame of that gene to permit propertranslation of mRNA, and stop codons.

The polynucleotide sequences encoding the CH2 or CH3 domain moleculescan be inserted into an expression vector including, but not limited toa plasmid, virus or other vehicle that can be manipulated to allowinsertion or incorporation of sequences and can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques known to those skilled in the art. Where thehost is prokaryotic, such as E. coli, competent cells which are capableof DNA uptake can be prepared from cells harvested after exponentialgrowth phase and subsequently treated by the CaCl₂ method usingprocedures well known in the art. Alternatively, MgCl₂ or RbC1 can beused. Transformation can also be performed after forming a protoplast ofthe host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the immunotoxin,antibody, or fragment thereof, and a second foreign DNA moleculeencoding a selectable phenotype, such as the herpes simplex thymidinekinase gene. Another method is to use a eukaryotic viral vector, such assimian virus 40 (SV40) or bovine papilloma virus, to transiently infector transform eukaryotic cells and express the protein (see for example,Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed.,1982). One of skill in the art can readily use an expression systemssuch as plasmids and vectors of use in producing proteins in cellsincluding higher eukaryotic cells such as the COS, CHO, HeLa and myelomacell lines.

Isolation and purification of recombinantly expressed polypeptide (suchas a CH2 domain molecule) can be carried out by conventional meansincluding preparative chromatography and immunological separations. Onceexpressed, the recombinantly expressed polypeptide can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.,1982). Substantially pure compositions of at least about 90 to 95%homogeneity are disclosed herein, and 98 to 99% or more homogeneity canbe used for pharmaceutical purposes. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

Methods for expression of a protein and/or refolding to an appropriateactive form, from bacteria such as E. coli have been described and arewell-known and are applicable to the antibodies disclosed herein. See,Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun,Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989 and Wardet al., Nature 341:544, 1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Renaturation can be accomplished by dilution (e.g., 100-fold) of thedenatured and reduced protein into refolding buffer. An exemplary bufferis 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione(GSSG), and 2 mM EDTA.

In addition to recombinant methods, the CH2 and CH3 domain moleculedisclosed herein can also be constructed in whole or in part usingstandard peptide synthesis. Solid phase synthesis of the polypeptides ofless than about 50 amino acids in length can be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2:Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield etal., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., SolidPhase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill.,1984. Proteins of greater length may be synthesized by condensation ofthe amino and carboxyl termini of shorter fragments. Methods of formingpeptide bonds by activation of a carboxyl terminal end (e.g., by the useof the coupling reagent N,N′-dicycylohexylcarbodiimide) are well knownin the art.

VII. Use of Antibody Constant Domain Molecules for Diagnosis orTreatment

CH2 and CH3 domain molecules have enormous potential for diagnosisand/or treatment of any of a number of diseases or conditions for whichan antibody is of use. For example, CH2 or CH3 domain molecules can beused for the treatment of cancer, infectious disease (such as viral,bacterial, fungal or parasitic infections), autoimmune disease,inflammatory disorders, or any other disease or condition for whichantibodies or their fragments can be used as therapeutic agents.

In some embodiments, the infectious disease caused by a virus, such as avirus from one of the following families: Retroviridae (for example,human immunodeficiency virus (HIV); human T-cell leukemia viruses(HTLV); Picornaviridae (for example, polio virus, hepatitis A virus;hepatitis C virus; enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses; foot-and-mouth disease virus); Calciviridae (such asstrains that cause gastroenteritis); Togaviridae (for example, equineencephalitis viruses, rubella viruses); Flaviridae (for example, dengueviruses; yellow fever viruses; West Nile virus; St. Louis encephalitisvirus; Japanese encephalitis virus; and other encephalitis viruses);Coronaviridae (for example, coronaviruses; severe acute respiratorysyndrome (SARS) virus; Rhabdoviridae (for example, vesicular stomatitisviruses, rabies viruses); Filoviridae (for example, Ebola viruses);Paramyxoviridae (for example, parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae (forexample, influenza viruses); Bunyaviridae (for example, Hantaan viruses;Sin Nombre virus, Rift Valley fever virus; bunya viruses, phlebovirusesand Nairo viruses); Arena viridae (hemorrhagic fever viruses; Machupovirus; Junin virus); Reoviridae (e.g., reoviruses, orbiviurses androtaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses; BK-virus); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV)-1 and HSV-2; cytomegalovirus (CMV);Epstein-Barr virus (EBV); varicella zoster virus (VZV); and other herpesviruses, including HSV-6); Poxyiridae (variola viruses, vacciniaviruses, pox viruses); and Iridoviridae (such as African swine fevervirus); Filoviridae (for example, Ebola virus; Marburg virus);Caliciviridae (for example, Norwalk viruses) and unclassified viruses(for example, the etiological agents of Spongiform encephalopathies, theagent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus); and astroviruses).

In other embodiments, the infectious disease is caused by a type ofbacteria, such as Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (such as. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus anthracis, corynebacteriumdiphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, or Actinomyces israelli.

In other embodiments, the infectious disease is caused by a fungus, suchas Cryptococcus neoformans, Histoplasma capsulatum, Coccidioidesimmitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candidaalbicans. In other embodiments, the infectious disease is caused by aparasite, such as Plasmodium falciparum or Toxoplasma gondii.

In some embodiments, the cancer is a solid tumor or a hematogenouscancer. In particular examples, the solid tumor is a sarcoma or acarcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, or another sarcoma, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, or aCNS tumor (such as a glioma, astrocytoma, medulloblastoma,craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma orretinoblastoma).

In some examples, the hematogenous cancer is a leukemia, such as anacute leukemia (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia); a chronic leukemia (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia ormyelodysplasia.

In some embodiments, the CH2 or CH3 domain molecule specifically binds atumor antigen. Tumor antigens are well known in the art and include, forexample, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin(β-HCG), alpha-fetoprotein (AFP), lectin-reactive AFP, (AFP-L3),thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase(hTERT), RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2,M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1,LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin and telomerase,prostate-carcinoma tumor antigen-1 (PCTA-1), melanoma-associated antigen(MAGE), ELF2M, neutrophil elastase, ephrinB2 and CD22. The CH2 or CH3domain molecules can also bind any cancer-related proteins, such IGF-I,IGF-II, IGR-IR or mesothelin. Additional tumor associated antigens areprovided below in Table 3.

TABLE 3 Exemplary tumors and their tumor antigens Tumor Tumor AssociatedTarget Antigens Acute myelogenous leukemia Wilms tumor 1 (WT1),preferentially expressed antigen of melanoma (PRAME), PR1, proteinase 3,elastase, cathepsin G Chronic myelogenous leukemia WT1, PRAME, PR1,proteinase 3, elastase, cathepsin G Myelodysplastic syndrome WT1, PRAME,PR1, proteinase 3, elastase, cathepsin G Acute lymphoblastic leukemiaPRAME Chronic lymphocytic leukemia Survivin Non-Hodgkin's lymphomaSurvivin Multiple myeloma New York esophageous 1 (NY-Eso1) Malignantmelanoma MAGE, MART, Tyrosinase, PRAME GP100 Breast cancer WT1,herceptin Lung cancer WT1 Prostate cancer Prostate-specific antigen(PSA) Colon cancer Carcinoembryonic antigen (CEA) Renal cell carcinoma(RCC) Fibroblast growth factor 5 (FGF-5)

In some embodiments, the autoimmune disease is rheumatoid arthritis,juvenile oligoarthritis, collagen-induced arthritis, adjuvant-inducedarthritis, Sjogren's syndrome, multiple sclerosis, experimentalautoimmune encephalomyelitis, inflammatory bowel disease (for example,Crohn's disease, ulcerative colitis), autoimmune gastric atrophy,pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obesediabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,sclerosing cholangitis, sclerosing sialadenitis, systemic lupuserythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia or pernicious anemia.

The wide utility of the CH2 and CH3 domain molecules is due at least inpart to their small size, which allows for efficient penetration intissues, including solid tumors and lymphoid tissue where HIVreplicates, and also permits efficient neutralization of viruses (forexample, HIV) that rapidly evolve to avoid neutralization byimmunoglobulins generated by the host immune system. Engineered CH2 orCH3 domain molecules are also useful for treatment due to theiramenability for creating high-affinity binding antibodies to any antigenof interest. Furthermore, as described herein, the CH2 or CH3 domainmolecules can further comprise an effector molecule with therapeuticproperties (such as, for example, a drug, enzyme or toxin).

As described herein, CH2 or CH3 domain molecules can be engineered tocomprise one or more CDRs from an antibody specific for a pathogen, suchas HIV. X5 is a neutralizing antibody specific for HIV-1 (Moulard et al.Proc. Natl. Acad. Sci. U.S.A. 99:6913-6918, 2002). The neutralizingactivity of X5 has been shown to significantly increase when convertedfrom a complete immunoglobulin (IgG1) or a Fab to a scFv antibody, whichcontains only the variable domains of the heavy and light chains(Labrijn et al. J. Virol. 77:10557-10565, 2003). It is believed thiseffect is due to the size-restricted access to the X5 epitope. CH2 andCH3 domain molecules are smaller than scFv antibodies, leading to thehypothesis that an engineered CH2 domain molecule (comprising one ormore X5 CDRs) would have enhanced neutralizing activity due to itsability to access the epitope.

CH2 and CH3 domain molecules are usually administered to a subject ascompositions comprising one or more pharmaceutically acceptablecarriers. Such carriers are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there is a wide variety ofsuitable formulations of pharmaceutical compositions of the presentdisclosure.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Administration can be accomplished by single or multiple doses. The doserequired will vary from subject to subject depending on the species,age, weight, general condition of the subject, the particular bleedingdisorder or episode being treated, the particular CH2 or CH3 domainmolecule being used and its mode of administration. An appropriate dosecan be determined by one of ordinary skill in the art using only routineexperimentation.

Provided herein are pharmaceutical compositions which include atherapeutically effective amount of an engineered CH2 or CH3 domainmolecule alone or in combination with a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers include, but are notlimited to, saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The carrier and composition can be sterile,and the formulation suits the mode of administration. The compositioncan also contain minor amounts of wetting or emulsifying agents, or pHbuffering agents. The composition can be a liquid solution, suspension,emulsion, tablet, pill, capsule, sustained release formulation, orpowder. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. Any of the common pharmaceuticalcarriers, such as sterile saline solution or sesame oil, can be used.The medium can also contain conventional pharmaceutical adjunctmaterials such as, for example, pharmaceutically acceptable salts toadjust the osmotic pressure, buffers, preservatives and the like. Othermedia that can be used with the compositions and methods provided hereinare normal saline and sesame oil.

VIII. Use of Antibody Constant Domain Molecules for Detection

Methods of determining the presence or absence of a polypeptide are wellknown in the art. For example, the specific binding agents, such as aCH2 domain molecule can be conjugated to other compounds including, butnot limited to, enzymes, magnetic beads, colloidal magnetic beads,haptens, fluorochromes, metal compounds, radioactive compounds or drugs.The CH2 or CH3 domain molecules can also be utilized in immunoassayssuch as but not limited to radioimmunoassays (RIAs), enzyme linkedimmunosorbent assays (ELISA), immunohistochemical assays, Western blotor immunoprecipitation assays. These assays are well known in the art(see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassayformats).

In one embodiment, a diagnostic kit comprising an immunoassay isprovided. Although the details of the immunoassays may vary with theparticular format employed, the method for detecting an antigen in abiological sample generally includes the steps of contacting thebiological sample with a CH2 or CH3 domain molecule which specificallyreacts, under immunologically reactive conditions, to the antigen ofinterest. The CH2 or CH3 domain molecule is allowed to specifically bindunder immunologically reactive conditions to form an immune complex, andthe presence of the immune complex (bound antigen) is detected directlyor indirectly.

The CH2 or CH3 domain molecules disclosed herein can also be used forfluorescence activated cell sorting (FACS). A FACS assay employs aplurality of color channels, low angle and obtuse light-scatteringdetection channels, and impedance channels, among other moresophisticated levels of detection, to separate or sort cells (see U.S.Pat. No. 5,061,620). FACS can be used to sort cells that are antigenpositive, by contacting the cells with an appropriately labeled CH2 orCH3 domain molecule. However, other techniques of differing efficacy maybe employed to purify and isolate desired populations of cells. Theseparation techniques employed should maximize the retention ofviability of the fraction of the cells to be collected. The particulartechnique employed will, of course, depend upon the efficiency ofseparation, cytotoxicity of the method, the ease and speed ofseparation, and what equipment and/or technical skill is required.

Additional separation procedures may include magnetic separation, usingCH2 or CH3 domain molecule-coated magnetic beads, affinitychromatography, cytotoxic agents, either joined to a CH2 or CH3 domainmolecule or used in conjunction with complement, and “panning,” whichutilizes an antibody, or CH2 or CH3 domain molecule, attached to a solidmatrix, or another convenient technique. The attachment of specificbinding agents to magnetic beads and other solid matrices, such asagarose beads, polystyrene beads, hollow fiber membranes and plasticPetri dishes, allow for direct separation. Cells that are bound by thespecific binding agent, such as a CH2 or CH3 domain molecule, can beremoved from the cell suspension by simply physically separating thesolid support from the cell suspension. The exact conditions andduration of incubation of the cells with the solid phase-linkedantibodies, or CH2 or CH3 domain molecules, will depend upon severalfactors specific to the system employed. The selection of appropriateconditions, however, is well known in the art.

Unbound cells then can be eluted or washed away with physiologic bufferafter sufficient time has been allowed for the cells expressing anantigen of interest to bind to the solid-phase linked binding agent. Thebound cells are then separated from the solid phase by any appropriatemethod, depending mainly upon the nature of the solid phase and theantibody or CH2 or CH3 domain molecule employed, and quantified usingmethods well known in the art. In one specific, non-limiting example,bound cells separated from the solid phase are quantified by FACS.

CH2 or CH3 domain molecules may be conjugated to biotin, which then canbe removed with avidin or streptavidin bound to a support, orfluorochromes, which can be used with FACS to enable cell separation andquantitation, as known in the art.

CH2 or CH3 domain molecules can be conjugated to other compoundsincluding, but not limited to, enzymes, paramagnetic beads, colloidalparamagnetic beads, haptens, fluorochromes, metal compounds, radioactivecompounds or drugs.

The enzymes that can be conjugated to the CH2 or CH3 domain moleculesinclude, but are not limited to, alkaline phosphatase, peroxidase,urease and β-galactosidase. The fluorochromes that can be conjugated tothe CH2 domain molecules include, but are not limited to, fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin,allophycocyanins and Texas Red. For additional fluorochromes that can beconjugated to antibodies see Haugland, R. P., Molecular Probes: Handbookof Fluorescent Probes and Research Chemicals (1992-1994). The metalcompounds that can be conjugated to the CH2 or CH3 domain moleculesinclude, but are not limited to, ferritin, colloidal gold, andparticularly, colloidal superparamagnetic beads. The haptens that can beconjugated to the CH2 or CH3 domain molecules include, but are notlimited to, biotin, digoxigenin, oxazalone, and nitrophenol. Additionalreagents are well known in the art.

IX. Effector Functions of Antibody Constant Domain molecules

Engineered CH2 or CH3 domains are capable of binding Fc receptors and/orcompliment-related molecules such as C1q, which allows for a variety ofeffector functions, including antibody-dependent cell-mediatedcytotoxicity (ADCC), complement dependent cytotoxicity (CDC),phagocytosis, opsonization and opsonophagocytosis. In some embodiments,the CH2 or CH3 domain molecules described herein comprise a binding sitefor one or more Fc receptors, thus enabling these molecules to mediatevarious effector functions (see Table 4 below). If effector functionsare not desirable, the Fc binding site(s) can be mutated to preventthese functions.

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, including a variety ofeffector functions and immunomodulatory signals. These interactions areinitiated through the binding of the Fc domain of antibodies or immunecomplexes to specialized cell surface receptors, Fc receptors. Eachmember of the Fc receptor family recognizes immunoglobulins of one ormore isotypes through a recognition domain on the Fc domain. Fcreceptors are defined by their specificity for immunoglobulin subtypes(for example, Fc receptors for IgG are referred to as FcγR) (U.S.Pre-Grant Publication No. 2006-0134709).

Fc receptors are glycoproteins found on the surface of some cells of theimmune system, including monocytes, macrophages, neutrophils,eosinophils, mast cells, natural killer cells, B cells and dendriticcells. Fc receptors exhibit a variety of cell expression patterns andeffector functions (see Table 4). Fc receptors allow immune cells tobind to antibodies that are attached to the surface of microbes ormicrobe infected cells, helping these cells to identify and eliminatemicrobial pathogens. The Fc receptors bind antibodies at their Fcregion, an interaction that activates the cell that possesses the Fcreceptor.

TABLE 4 Cell Distribution and Effector Functions of Fc ReceptorsReceptor name Cell distribution Effector function FcγRI MacrophagesPhagocytosis (CD64) Neutrophils Cell activation Eosinophils Activationof respiratory burst Dendritic cells Induction of microbe killingFcγRIIA Macrophages Phagocytosis (CD32) Neutrophils Degranulation(eosinophils) Eosinophils Platelets Langerhans cells FcγRIIB1 B Cells Nophagocytosis (CD32) Mast cells Inhibition of cell activity FcγRIIB2Macrophages Phagocytosis (CD32) Neutrophils Inhibition of cell activityEosinophils FcγRIIIA NK cells Induction of ADCC (CD16a) FcγRIIIBEosinophils Induction of microbe killing (CD16b) Macrophages NeutrophilsMast cells Follicular dendritic cells FcεRI Mast cells DegranulationEosinophils Basophils Langerhans cells FcεRII B cells Possible adhesionmolecule (CD23) Eosinophils Langerhans cells FcαRI MonocytesPhagocytosis (CD89) Macrophages Induction of microbe killing NeutrophilsEosinophils Fcα/μR B cells Endocytosis Mesangial cells Induction ofmicrobe killing Macrophages FcRn Monocytes Transfers IgG from a motherto Macrophages fetus through the placenta Dendritic cells Transfers IgGfrom a mother to Epithelial cells infant in milk Endothelial cellsProtects IgG from degradation Hepatocytes

Activation of phagocytes is the most common function attributed to Fcreceptors. For example, macrophages begin to ingest and kill an IgGcoated pathogen by phagocytosis following engagement of their Fcγreceptors. Another process involving Fc receptors is calledantibody-dependent cell-mediated cytotoxicity (ADCC). During ADCC,FcγRIII receptors on the surface of natural killer (NK) cells stimulatethe NK cells to release cytotoxic molecules from their granules to killantibody covered target cells. However, FcεRI has a different function.FcεRI is the Fc receptor on granulocytes that is involved in allergicreactions and defense against parasitic infections. When an appropriateallergic antigen or parasite is present, the cross-linking of a leasttwo of IgE molecules and their Fc receptors on the surface of agranulocyte will trigger the cell to rapidly release preformed mediatorsfrom its granules.

In addition, the Fc domains of IgG and IgM antibodies are capable ofbinding C1q, a component of the classical pathway of complementactivation. When IgG or IgM antibodies are bound to the surface of apathogen, C1q is capable of binding their Fc regions, which initiatesthe complement cascade, ultimately resulting in the recruitment ofinflammatory cells and the opsonization and killing of pathogens.

To further provide functionality to the CH2 or CH3 domain molecules,effector molecules (for example, therapeutic, diagnostic, or detectionmoieties) can be linked to a CH2 or CH3 domain molecule using any numberof means known to those of skill in the art. Exemplary effectormolecules include, but are not limited to, radiolabels, fluorescentmarkers, or toxins. Both covalent and noncovalent attachment means canbe used. The procedure for attaching an effector molecule to an antibodyvaries according to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups; for example,carboxylic acid (COOH), free amine (—NH₂) or sulfhydryl (—SH) groups,which are available for reaction with a suitable functional group on anantibody to result in the binding of the effector molecule.Alternatively, the antibody is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody to the effector molecule. Thelinker is capable of forming covalent bonds to both the antibody and tothe effector molecule. Suitable linkers are well known to those of skillin the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (such as through a disulfide linkage to cysteine) or to thealpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (for example, enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies, one skilled in the art will beable to determine a suitable method for attaching a given agent to a CH2or CH3 domain molecule.

Therapeutic agents include various drugs such as vinblastine, daunomycinand the like, and effector molecules such as cytotoxins such as nativeor modified Pseudomonas exotoxin or Diphtheria toxin, encapsulatingagents, (such as, liposomes) which themselves contain pharmacologicalcompositions, target moieties and ligands. The choice of a particulartherapeutic agent depends on the particular target molecule or cell andthe biological effect desired to be evoked. Thus, for example, thetherapeutic agent may be an effector molecule that is cytotoxic which isused to bring about the death of a particular target cell. Conversely,where it is merely desired to invoke a non-lethal biological response, atherapeutic agent can be conjugated to a non-lethal pharmacologicalagent or a liposome containing a non-lethal pharmacological agent.

Toxins can be employed with a CH2 or CH3 domain molecule which is of useas an immunotoxin. Exemplary toxins include Pseudomonas exotoxin (PE),ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin,ribonuclease, saporin, and calicheamicin, as well as botulinum toxins Athrough F. These toxins are well known in the art and many are readilyavailable from commercial sources (for example, Sigma Chemical Company,St. Louis, Mo.).

Diphtheria toxin is isolated from Corynebacterium diphtheriae.Typically, diphtheria toxin for use in immunotoxins is mutated to reduceor to eliminate non-specific toxicity. A mutant known as CRM107, whichhas full enzymatic activity but markedly reduced non-specific toxicity,has been known since the 1970's (Laird and Groman, J. Virol. 19:220,1976), and has been used in human clinical trials. See, U.S. Pat. No.5,792,458 and U.S. Pat. No. 5,208,021. As used herein, the term“diphtheria toxin” refers as appropriate to native diphtheria toxin orto diphtheria toxin that retains enzymatic activity but which has beenmodified to reduce non-specific toxicity.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). The term“ricin” also references toxic variants thereof. For example, see, U.S.Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Ricinus communisagglutinin (RCA) occurs in two forms designated RCA₆₀ and RCA₁₂₀according to their molecular weights of approximately 65 and 120 kD,respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543,1972). The A chain is responsible for inactivating protein synthesis andkilling cells. The B chain binds ricin to cell-surface galactoseresidues and facilitates transport of the A chain into the cytosol(Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

Ribonucleases have also been conjugated to targeting molecules for useas immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999).Exemplary ribotoxins such as α-sarcin and restrictocin are discussed in,for example, Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra,Biochem 345 Pt 2:247-54, 2000. Calicheamicins were first isolated fromMicromonospora echinospora and are members of the enediyne antitumorantibiotic family that cause double strand breaks in DNA that lead toapoptosis (see, e.g., Lee et al., J. Antibiot 42:1070-87. 1989). Thedrug is the toxic moiety of an immunotoxin in clinical trials (see, forexample, Gillespie et al., Ann Oncol 11:735-41, 2000).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B chain (abrin-b)binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem.52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 Generation of a Library of Antibody CH2 Domains withLoops Containing Amino Acid Residues Randomly Mutated to any of the FourResidues, Y, S, A or D

In this example, mutated CH2 domains were constructed in which loop 1was replaced with 10 randomly arranged Y, S, A or D residues, plus anadditional G at the C-terminal end of the loop. Similarly, loop 3 wasreplaced with 6 randomly arranged Y, S, A or D residues, plus anadditional G at the C-terminal end of the loop. The DNA library isgenerated in three stages.

First, the CH2 DNA is used for generation of two fragments, fragment 1and fragment 2, containing mutated loop 1 and loop 2, respectively.Fragment 1 is generated by PCR amplification using an N-terminal primer(5′ GCA CTG GCT GGT TTC GCT ACC GT GGCC CAGGC GGCC GCA CCT GAA CTC CTG3′; SEQ ID NO: 6) and a loop 1 reverse primer (5′ CAC GTA CCA GTT GAACTT GCC AKM AKM AKM AKM AKM AKM AKM AKM AKM AKM CAC CAC CAC GCA TGT GAC3′; SEQ ID NO: 7), where K=G or T, and M=A or C. Fragment 2 is generatedby using a loop 1 forward primer (5′ AAG TTC AAC TGG TAC GTG 3′; SEQ IDNO: 8) and a loop 3 reverse primer (5′ GAT GGT TTT CTC GAT GGG GCC AKMAKM AKM AKM AKM AKM GTT GGA GAC CTT GCA CTT G 3′; SEQ ID NO: 9).

Second, the two fragments are joined by the use of splicing byoverlapping extension (SOE) PCR. During the second step of the SOE PCR,a C-terminal primer (5′ GGT GCA GAA GAT GGT GGT GGCC GGCCT GGCC TTT GGCTTT GGA GAT GGT TTT CTC GAT G 3′; SEQ ID NO: 10) is used in addition tothe N-terminal primer to introduce the restriction site Sfi1 on bothends of the DNA which is needed for the next stage of cloning.

Third, the amplified mutated CH2 fragments are digested with Sfi1 andligated into a phagemid vector digested with the same enzyme. Theproduct of ligation is desalted by washing three times with doubledistilled water using Amicon Ultra-4 centricon before transformation ofTG1 cells by electroporation.

Sequences of 20 randomly selected clones from transformed TG1 cells areshown below (Table 5), demonstrating successful generation of CH2mutants with randomized loops 1 and 3 by four residues, Y, S, D and A.

TABLE 5 Fragments of mutant CH2 sequences with randomized loops 1 and 3(X2-22 denote names of clones) Loop 1 x9PEVTCVVV YYDSAAAYAY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 11) x14PEVTCVVV YYSASAAASA GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 12) x13PEVTCVVV YDSDYASSDD GKFNWYVDG VEVHNAKTKP RKEQYNSTYR (SEQ ID NO: 13) x15PEVTCVVV AYSDDAAAYD GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 14) x10PEVTCVVV DADDDYYYYY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 15) x2PEVTCVVV DDAYYDADYYY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 16) x11PEVTCVVV DAAYDYSY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 17) x19PEVTCVVV DYDSDDAYAD GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 18) x16PEVTCVVV SYYDSDSYSA GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 19) x4PEVTCVVV DDAYADDASA GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 20) x17PEVTCVVV SYYSDSDYDD GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 21) x12PEVTCVVV DDDSYYSYDD GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 22) x22PEVTCVVV YDASDYADAY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 23) x8PEVTCVVV ADAAAYAYAD GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 24) x7PEVTCVVV ASDSSDDYD GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 25) x5PEVTCVVV AAAAADADYY SKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 26) x20PEVTCVVV YDDAAYADDY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 27) x21PEVTCVVV SADASDYD GKFNWYVDG VEVHNADTKP REEQYNSTYR (SEQ ID NO: 28) x23PEVTCVVV DDDAADAYYY GKFNWYVDG VEVHNAKTKP REEQYNSTYR (SEQ ID NO: 29) x3PEVTCVVV YDSDDDYDYA GKFCWYVDG VEVHNAKTKP REEHYNSTYR (SEQ ID NO: 30)Loop 3 x9 VVSVLTVLHQ DWLNGKEYKC KVSN AASAYS GPIEKTISKA K (SEQ ID NO: 31)x14 VVSVLTVLHQ DWLNGKEYKC KVSN ADDADA GPIEKTISKA K (SEQ ID NO: 32) x13VVSVLTVLHQ DWLNGKEYKC KVSN AADAYA GPIEKTISKA K (SEQ ID NO: 33) x15VVSVLTVLHQ DWLNGKEYKC KVSN AADYSD GPIEKTISKA K (SEQ ID NO: 34) x10VVSVLTVLHQ DWLNGKEYKC KVSN AADAAD GPIEKTISKA K (SEQ ID NO: 35) x2VVSVLTVLHQ DWLNGKEYKC KVSN DASASS GPIEKTISKA K (SEQ ID NO: 36) x11VVSVLTVLHQ DWLNGKEYKC KVSN DDYAAS GPIEKTISKA K (SEQ ID NO: 37) x19VVSVLTVLHQ DWLNGKEYKC KVSN DAYASD GPIEKTISKA K (SEQ ID NO: 38) x16VVSVLTVLHQ DWLNGKEYKC KVSN DADDAS GPIEKTISKA K (SEQ ID NO: 39) x4VVSVLTVLHQ DWLNGKEYKC KVSN AADDDS GPIEKTISKA K (SEQ ID NO: 40) x17VVSVLTVLHQ DWLNGKEYKC KVSN ADAYAY GPIEKTISKA K (SEQ ID NO: 41) x12VVSVLTVLHQ DWLNGKEYKC KVSN ADDYDY GPIEKTISKA K (SEQ ID NO: 42) x22VVSVLTVLHQ DWLNGKEYKC KVSN YSDSAA GPIEKTISKA K (SEQ ID NO: 43) x8VVSVLTVLHQ DWLNGKEYKC KVSN YAASAY GPIEKTISKA K (SEQ ID NO: 44) x7VVSVLTVLHQ DWLNGKEYKC KVSN YDDDAD GPIEKTISKA K (SEQ ID NO: 45) x5VVSVLTVLHQ DWLNGKEYKC KVSN YYDYDY GPIEKTISKA K (SEQ ID NO: 46) x20VVSVLTVLHH DWMNGKEYKC EVSN DADSAD GPIKKTISKA K (SEQ ID NO: 47) x21VVSVLTVLHH DWLNGEEYKC KVSN DASDDA GPIEKTIS.A K (SEQ ID NO: 48) x23VVSVLTVLHQ DWLNGKEYKC KVSN ADDAYA GPIEKTISKA K (SEQ ID NO: 49) x3VVSVLTVLHH YWMNGEDYKC EVSN DSYSDD GPIKKTISKA K (SEQ ID NO: 50)

Example 2 Engraftment of CDR3s from Human Antibodies into CH2 Scaffold

In this example human VH CDR3s (H3s) from an antibody library areengrafted into CH2 by replacing loops A-B and E-F. First, the loop A-Bis replaced by H3s using five PCRs. The first two PCRs generate two CH2fragments without the loop A-B by using the following primers: forfragment 1—forward 1 primer (5′ TAG CGA TTC GCT ACC GTG GCC CAG GCG GCCCCT GAA CTC CTG GGG GGA CC 3′; SEQ ID NO; 51) and reverse 1 primer (5′TCC CCC CAG GAG TTC AGG TGC 3′; SEQ ID NO; 52), for fragment 2—forward 2primer (5′ TGC GTG GTG GTG GAC GTG AGC 3′; SEQ ID NO: 53) and reverse 2primer (5′ TAG GCA TGC ATC TGC ATG GTG GCC GGC CTG GCC TTT GGC TTT GGAGAT GGT TTT CTC GAT GG 3′; SEQ ID NO: 54). The forward 1 and the reverse2 primers contain the restriction site for Sfi/which is required at theN- and C-termini in the final product. The reverse 1 and forward 2primers contain end sequences needed for a subsequent SOE PCR. The thirdPCR uses as a template an antibody VH library and two mixtures of threeprimers each, designed to amplify diverse H3s. The mixture of forwardprimers contains H3 forward primer 1: 5′ GAA CTC CTG GGG GGA CCG GCY AYRTAT TAC TGT GYG 3′ (SEQ ID NO: 55), H3 forward primer 2: 5′ GAA CTC CTGGGG GGA CCG GCY TTR TAT TAC TGT GYG 3′ (SEQ ID NO: 56), and H3 forwardprimer 3: 5′ GAA CTC CTG GGG GGA CCG GCY GTR TAT TAC TGT GYG 3′ (SEQ IDNO: 57). The mixture of reverse primers contains H3 reverse primer 1: 5′GCT CAC GTC CAC CAC CAC GCA GGT GCC CTG GCC CCA 3′ (SEQ ID NO: 58), H3reverse primer 2: 5′ GCT CAC GTC CAC CAC CAC GCA GGT GCC ACG GCC CCA 3′(SEQ ID NO: 59), and H3 reverse primer 3: 5′ GCT CAC GTC CAC CAC CAC GCAGGT GCC AYG GCC CCA 3′ (SEQ ID NO: 60). It generates a mixture offragments containing H3s with end sequences designed to overlap with therespective end sequences of the reverse 1 and forward 2 primers. The twoCH2 fragments and the H3 containing fragments are used as primers andtemplates in a SOE PCR to generate a fragment where loop AB is replacedby H3s. This mixture of fragments is amplified by using the forward 1primer and the reverse 2 primers. The amplified fragments are digestedwith Sfi1 and ligated into a phagemid vector (pComb3X or pZUD) digestedwith the same enzyme. The product of ligation is desalted by washingthree times with double distilled water using Amicon Ultra-4 centriconbefore transformation of TG1 cells by electroporation.

A similar procedure can be used for replacement of loop E-F, except thatfor amplification of fragment 1, instead of reverse primer 1 anotherprimer-reverse primer 12 (5′ GTA CGT GCT GTT GTA CTG CTC 3′; SEQ ID NO:61) is used; for amplification of fragment 2—instead of forward primer 2another primer—forward primer 22 (5′ AAG GTC TCC AAC AAA GCC CTC 3′; SEQID NO: 62) is used; and for amplification of the H3s, the H3 primers aredifferent. In this case, the mixture of forward primers contains H3forward primer 12: 5′ GAG CAG TAC AAC AGC ACG TAC GCA GCY AYR TAT TACTGT GYG 3′ (SEQ ID NO: 63), H3 forward primer 22: 5′ GAG CAG TAC AAC AGCACG TAC GCA GCY TTR TAT TAC TGT GYG 3′ (SEQ ID NO: 64), and H3 forwardprimer 32: 5′ GAG CAG TAC AAC AGC ACG TAC GCA GCY GTR TAT TAC TGT GYG 3′(SEQ ID NO: 65). The mixture of reverse primers in this case contains H3reverse primer 12: 5′ GAG GGC TTT GTT GGA GAC CTT GGT TCC CTG GCC CCA 3′(SEQ ID NO: 66), H3 reverse primer 22: GAG GGC TTT GTT GGA GAC CTT GGTGCC ACG GCC CCA 3′ (SEQ ID NO: 67), and H3 reverse primer 32: 5′ GAG GGCTTT GTT GGA GAC CTT GGT GCC AYG GCC CCA 3′ (SEQ ID NO: 68). Finally,both loops, A-B and E-F, can be replaced with VH H3s. In this case.following replacement of loop A-B by H3s, loop E-F is replaced in theresulting fragments by H3s which are randomly recombined.

Sequences of 19 randomly selected clones from transformed TG1 cells withboth loops replaced by H3s are shown below (Table 6) suggestingsuccessful grafting of H3s. FIG. 4 shows protein expression for severalof these clones. The positions of the bands of the mutant molecules areindicated with an arrow.

TABLE 6 Fragments with grafted H3s#2-38 (SEQ ID NOs: 69-87, respectively) denote names of clones            H3                                  H3  #2:AVYYCV.KVPVGY............WGRGT & AVYYCA.DVEASSPADFGY....WGRGT  #3:AMYYCA.RDHGVDTAMAGPWFDY..WGRGT & AVYYCV.RGTGWELLVIDC....WGRGT  #6:AVYYCA.RGSSGWGWFDP.......WGQGT & ATYYCA.RDRGY...........WGRGT  #8:AVYYCA.RRMPEGDSSGTSYYFDY.WGQGT & ALYYCA.REEKGDYDY.......WGQGT #13:AMYYCA.IHSFDY............WGQGT & AVYYCA.KVLSGWFDHYFDS...WGQGT #15:AVYYCA.RDRVPDGVWSADS.....WGQGT & AVYYCA.SKPPVSNWFDP.....WGQGT #16:AIYYCV.KAGYNFDAFDH.......WGRGT & AMYYCA.GDTAMVIFDY......WGQGT #17:ATYYCA.SGSSGCSDY.........WGQGT & ATYYCA.RGGYSSGWYHWYFDL.WGRGT #22:AVYYCA.ASVGAPSDFDY.......WGQGT & ATYYCA.TTPDSNYGY.......WGQGT #23:ALYYCA.KGQYGDHDY.........WGQGT & AVYYCA.KEEEGAVLG.......WGRGT #25:ATYYCA.REGTVVTPYFVY......WGQGT & AVYYCA.MGGHGSGSYLSGY...WGQGT #26:AVYYCA.RERYGALDY.........WGRGT & AVYYCA.GGLLHEGSGY......WGQGT #28:AIYYCA.ARGQGNSWWFDP......WGQGT & AIYYCA.TQVGHGD.........WGQGT #30:ALYYCA.RAYSAYQYSFDS......WGRGT & AVYYCA.RREYNWNHNWFDP...WGQGT #31:ATYYCA.RRGDDYGDYFFDY.....WGQGT & AIYYCA.RSRGSSFDY.......WGQGT #33:AMYYCA.RDLYSNYVDY........WGQGT & AVYYCA.RGPWQQLVNWFDP...WGQGT #34:ATYYCA.SLTGTTSY..........WGQGT & ALYYCA.RATWGYQFDC......WGQGT #36:AIYYCA.RESSSSFDY.........WGQGT & AVYYCA.RMSGGRWIFDH.....WGQGT #38:AVYYCA.RGWELDY...........WGQGT & AVYYCA.KTGQFDY.........WGQGT

Example 3 Engineering and Characterization of Stabilized CH2 Mutants

In this example, two mutants of CH2 are identified that exhibit anincreased stability compared to the parental wild type CH2. Because theCH2 framework is already stabilized by internal disulfide bond betweenstrands B and F, it was hypothesized that an additional disulfide bondbetween other strands could provide an overall increase in the CH2stability. Several positions in strand A and G were mutated, of whichone resulted in a very stable mutant CH2, designated as m01, where L (inthe sequence GPSVFLFPPKPKDTL (SEQ ID NO: 88)) and the first K (in thesequence EKTISKAK (SEQ ID NO: 89)) were mutated to C. Another mutant,designated m02, where V (in GPSVFLFPPKPKDTL (SEQ ID NO; 90)) and thefirst K in EKTISKAK (SEQ ID NO: 91) were mutated to C, exhibited anincrease in stability compared to the parental CH2, but lower than thatof m01.

Materials and Methods

Cloning, Expression and Purification of CH2 Domains.

Human γ1 CH2 was cloned in bacterial expression vectors and used fortransformation of Escherichia coli strain HB2151 cells which were grownat 37° C. in SB medium to an optical density of OD₆₀₀˜0.6-0.8.Expression was induced with 1 mM IPTG at 37° C. for 12-16 hrs. Bacterialcells were harvested and re-suspended in Buffer A (50 mM Tris.Cl, 450 mMNaCl, pH 8.0) at 1:10 (volume of Buffer A: culture volume). Polymyxin Bsulfate (Sigma-Aldrich, MO) (0.5 mu/ml) was added to the suspension(1:1000 volume of polymyxin B sulfate: culture volume). The cell lysatewas subsequently clarified by centrifugation at 15,000 rpm for 45 min at4° C. and tested for expression by SDS-PAGE and Western. The clarifiedsupernatant was purified by using 1 ml HiTrap Chelating HP Ni-NTA column(GE Healthcare, N.J.). After elution with Buffer B (50 mM Tris.Cl, 450mM NaCl, 200 mM Imidazole, pH 8.0), the Imidazole was removed by AmiconUltra—15 Centrifugal Filter Devices (MILLIPORE, MA) and the purifiedproteins were kept in Buffer A or PBS (9.0 g/L NaCl, 144 mg/L KH₂PO₄,795 mg/L Na₂HPO₄, pH 7.4). The proteins were checked for purity bySDS-PAGE and their concentrations were determined by measuring the UVabsorbance.

CH2 Mutant Design and Plasmid Construction.

To design the CH2 mutants the Fc crystal structure was used. Fivemutants, V10/E103 to C10/C103, F11/K104 to C11/C104, L12/T105 toC11/C105, L12/K104 to C12/C104, and V10/K104 to C10/C104, were selectedfor characterization by analyzing the structure with the computerprogram VMD 18.6 (Humphrey et al., J Mol. Graph. 14:33-38, 1996). Theywere made by PCR-based site-directed mutagenesis and cloned intobacterial expression vectors. The clones were verified by directsequencing and used for transformation of the Escherichia coli strainHB2151. The mutants were expressed and purified similarly to the wildtype CH2.

Size Exclusion Chromatography.

Purified CH2, CH2 m01 and CH2 m02 were loaded into the Hiload 26/60Superdex 75 HR 10/30 column (GE Healthcare, NJ) running on AKTA BASICpH/C chromatography system (GE Healthcare, NJ) to assess oligomerformation. Buffer A was selected as mobile phase. A gel-filtrationstandard consisting of aldolase (158 kD), bovine serum albumin (67 kDa),ovalbumin (44 kDa), chymotrypsinogen A (25 kD) and ribonuclease A (17kDa) was used to define the molecular weight of CH2, CH2 m01 and CH2m02.

Determination of Disulfide Bonds by Mass Spectrometry.

The total number of disulfide bonds in purified CH2, CH2 m01 and CH2 m02was determined through Voyager 4700 MALDI-TOF/TOF mass spectrometry)(Applied Biosystems, CA) by comparing the molecular masses after (A)reduction and alkylation of all SH groups and (B) alkylation of theoriginal free SH groups without reduction of disulfide bonds. Reductionwas carried out with TCEP, and alkylation was performed withiodoacetamide.

Circular Dichroism (CD).

The secondary structure of CH2, CH2 m01 and CH2 m02 were determined bycircular dichroism (CD) spectroscopy. The purified proteins weredissolved in PBS at the final concentration of 0.49 mg/ml, and the CDspectra were recorded on AVIV Model 202 CD Spectrometer (AvivBiomedical, NJ). Wavelength spectra were recorded at 25° C. using a 0.1cm path-length cuvette for native structure measurements. Thermodynamicstability was measured at 216 nm by recording the CD signal in thetemperature range of 25-90° C. with heating rate 1° C./min. Afterheating, wavelength spectra were recorded at 90° C. For evaluation ofthe refolding, all the samples were kept at 4° C. overnight and measuredagain at 25° C. The temperature was recorded with an external probesensor and the temperature inside the microcuvette was calculated bycalibration—it was about 2-3° C. (range from 1.9° C. to 3.8° C. fortemperatures from 20° C. to 80° C.) lower that the one measured by theexternal sensor.

Differential Scanning Calorimetry (DSC).

The thermal stabilities of CH2, CH2 m01 and CH2 m02 were furthermonitored with a VP-DSC MicroCalorimeter (MicroCal, Northampton, Mass.).The concentrations of three proteins were 1.5 mg/ml in PBS (pH 7.4). Theheating rate employed was 1° C./min and the scanning was performed from25 to 100° C.

Spectrofluorometry.

The intrinsic fluorescence of CH2, m01 and m02 were recorded on aFluorometer Fluoromax-3 (HORIBA Jobin Yvon, NJ). Intrinsic fluorescencemeasurements were performed using a protein concentration of 10 μg/mlwith excitation wavelength at 280 nm, and emission spectra recorded from320 to 370 nm at 25° C. Buffer A in the presence of urea from 0 to 8 mMwas used. With all samples, fluorescence spectra were corrected for thebackground fluorescence of the solution (buffer+denaturant).Fluorescence intensity at 340 nm was used for unfolding evaluation.

Nuclear Magnetic Resonance (NMR).

For the NMR experiments E. coli was first grown in 2×YT. Single colonywas inoculated in 3 mL 2×YT for about 3 hrs, then turbidity was checkedand bacteria transferred to 1 liter 2×YT medium for further growth at37° C. until OD₆₀₀˜0.8-0.9 was reached. The cell culture was thencentrifuged to remove the 2×YT medium and replaced it with a M9 minimummedium with ¹⁵N NH₄Cl and ¹³C glucose as sole ¹⁵N and ¹³C sources,respectively (17). The cells were incubated at 30° C. overnight, andinduced with 1 mM IPTG. Harvested cells were suspended in TES buffer (10mL buffer for 1 L of culture) for 1 h on ice. Osmotic shock to releaseperiplasmic proteins was induced by adding 1.5 volume TES/5 on ice for 4hrs. The supernatant was then dialyzed in a dialysis buffer (50 mMTris.Cl, 0.5 M NaCl) over night at 4° C. The protein was purified by themethod described above for an initial purification. Fractions containinga significant amount of the protein were then loaded on Sephacryl S-200column (GE Healthcare, NJ) for further purification. The separatedfractions samples were collected in Buffer A.

NMR experiments were performed in 40 mM Tris.Cl buffer at pH 7.8containing 64 mM NaCl in 95% H₂O/5% D₂O and a sample volume ofapproximately 300 μl in a 5-mm Shigemi tube (Shigemi Inc, PA) with aprotein concentration of 0.5-0.8 mM at 25° C. NMR experiments wereconducted using a Bruker Avance 600 MHz instrument which is equippedwith a cryogenic probe (Bruker Instruments, MA). Water-flip backsequences were used for ¹H-¹⁵N HSQC and {¹H}-¹⁵N NOE experiments tominimize exchange between amide protons and water protons (Grzesiek andBax, J. Am. Chem. Soc. 115:12593, 1994). ¹H-¹⁵N HSQC spectra wererecorded with 1024 complex points for an acquisition dimension with aspectral width of 8012 Hz, and 256 complex points for an indirect (t₁)dimension. {¹H}-¹⁵N NOE experiments were conducted with the similarnumber of points by recording two sets of spectra, with and withoutproton saturation at 3 and 4 second repetition delays, respectively(Gong and Ishima, J. Biomol. NMR 37:147-157, 2007). Uncertainties of theNOE values were estimated from r.m.s.d. noise of the two spectra andpeak heights.

Signal assignments were performed based on HNCA, CBCACONH, CCONHexperiments for CH2 domain, and HNCACB and CBCACONH and ¹³C, ¹⁵Nsimultaneous evolution NOESY for CH2 m01 domain (Kay et al., J. Magn.Reson. 89:496-514, 1990; Muhandiram and Kay, J. Magn. Res. Series B.103:203-216, 1994). NMR data were processed and analyzed using thenmrPipe (Delaglio et al., J. Biomol. NMR 6:277-293, 1995; Masse andKeller, J. Magn. Reson. 174:133-151, 2005). To color significance ofchemical shift changes on CH2 backbone structure, a normalized chemicalshift changes, δ_(norm)√{square root over((δ_(Cα))+(γ_(Cα)/γ_(N))²+(δ_(N))₂(δ_(N))²)}{square root over((δ_(Cα))+(γ_(Cα)/γ_(N))²+(δ_(N))₂(δ_(N))²)}{square root over((δ_(Cα))+(γ_(Cα)/γ_(N))²+(δ_(N))₂(δ_(N))²)}{square root over((δ_(Cα))+(γ_(Cα)/γ_(N))²+(δ_(N))₂(δ_(N))²)}, its average, and standarddeviation (s.d.) were calculated, and are grouped to four classes:δ_(norm)>3.0 (red), 3.0>δ_(norm)>2.0 (orange), 2.0>δ_(norm)>1.0(yellow), and (4) δ_(norm)<1 (blue).

Results

Isolated, Unglycosylated Human γ1 CH2 Domain is Relatively Stable.

Human γ1 heavy chain CH2 (FIG. 5A) was cloned in a bacterial expressionvector, expressed and purified as described in above. Human γ1 CH2expresses at high levels as soluble protein (more than 10 mg per literof bacterial culture) and is highly soluble (more than 10 mg/ml). It ismonomeric in PBS at pH 7.4 as determined by size exclusionchromatography (FIG. 5B) (Prabakaran et al., Acta Crystallogr. B.64:1062-1067, 2008). SDS-PAGE of human γ1 CH2 revealed an apparentmolecular weight (MW) of about 14-15 kDa, which is close to thecalculated MW (14.7 kDa, including the His and FLAG tags). As expected,it is much smaller than the MWs of scFv, Fab and IgG1 (FIG. 5C).

Previously, it has been found that an isolated unglycosylated murine CH2domain is relatively unstable at physiologically relevant temperatures(T_(m)=41° C. as measured by circular dichroism (CD) (Feige et al., J.Mol. Biol. 344:107-118, 2004). The sequence of human CH2 differs fromthat of the murine one which could lead to different stabilities (FIG.5A). To test the thermodynamic stability of human γ1 CH2, both CD anddifferential scanning calorimetry (DSC) were used. As measured by CD,the secondary structure of CH2 consisted of beta strands at 25° C. TheCH2 unfolding started at about 42° C. and was completed at about 62° C.(FIG. 6A) with a calculated Tm of 54.1±1.2° C. (FIG. 6A), The unfoldingwas reversible (FIG. 6A). Similar results were obtained by DSC(T_(m)=55.4° C., FIG. 6B). Thus the human γ1 CH2 is significantly morestable than its murine counterpart.

Design and Generation of Engineered Human γ1 CH2 Domains with anAdditional Disulfide Bond.

To further improve the stability of human CH2, an additional disulfidebond was engineered between the N-terminal strand A and the C-terminalstrand G. It was reasoned that constraining the degrees of freedom ofthese two strands could lead to a decrease in the extent of unfolding.The mutants were initially designed based on the crystal structure ofCH2 in an intact Fc which is very similar to the crystal structure ofisolated CH2 which was recently reported although there are certaindifferences in some loops and at the termini (Prabakaran et al., ActaCrystallogr. B. 64:1062-1067, 2008). Based on the distance between two Cα-carbons in proteins with known structure (Dani et al., Protein Eng.16:187-193, 2003; Pellequer and Chen, Proteins 65:192-202, 2006) and theorientation of the bonds, five amino acid pairs were selected: V10/E103,F11/K104, L12/T105, L12/K104 and V10/K104 (the numbering starts with1:Ala, corresponding to number 231 in the γ1 heavy chain) (FIG. 5A; SEQID NO: 5), which were substituted by Cys. Two mutants (L12/K104 toC12/C104, distance between the C^(α)s in L12 and K104=6.53 Å, andV10/K104 to C10/C104, distance between the C^(α)s in V10 and K104=7.25Å) (FIG. 7), designated m01 and m02, respectively, were highly solubleand expressed at levels comparable or higher than CH2 (FIG. 8).

The existence of an additional disulfide bond was confirmed by massspectrometry. The number of disulfide bonds in CH2 was one, and inmutants m01 and m02 it was two, as expected (Table 7). These mutantswere selected for further characterization.

TABLE 7 Number of disulfide bonds determined by mass spectrometryDenatured Reduced Reduced/Alkylate Alkylated Number Protein Intact (Da)(D) (Da) (R) (Da) (R/A) (Da) (A) (Da) N_(cys) N_(SH) of -s-s- CH214707.3607 14714.5977 14710.9160 14822.6719 14708.5791 2 0 1 CH214674.3447 14677.7539 14676.3398 14899.9238 14669.0400 4 0 2 m01 CH214688.9561 14686.2461 14695.6543 14901.8076 14686.1230 4 0 2 m02 N_(cys)= (M_(R/A))/57 N_(SH) = (M_(A) − M_(D))/57 Number of disulfide bond(-s-s-) = (N_(cys) − N_(SH))/2

m01 and m02 are Significantly More Stable than CH2.

The thermodynamic stability of m01, m02 and CH2 was measured by CD andDSC, and their stability against chemical agents was determined by usingurea and spectrofluorimetry. In all cases, the two mutants were muchmore stable than CH2 (FIG. 9). The CD spectra of CH2, m01 and m02 showedthat they had high β-sheet content at 25° C. (FIG. 9A and 9B). Theβ-sheet structure was gradually disrupted as the temperature increased(FIG. 9C). At 90° C., the structure was in an unfolded state (FIGS. 9Aand 9B). The sigmoidal curve was fitted by a two-state model as alsopreviously reported (Feige et al., J. Mol. Biol. 344:107-118, 2004).Notably, 50% unfolding of m01 and m02 occurred at temperatures(Tm=73.8±1.7° C. and 65.3±0.6° C., respectively) that were significantlyhigher than that of native CH2 (54.1±1.2° C.) (FIG. 9C). CH2 and m01refolded reversibly; however, m02 only partially re-folded (FIG. 9A andFIG. 6A versus FIG. 9B).

Similar results were obtained by DSC. The melting temperatures of m01and m02 were much higher than that of native CH2, which also increasedabout 20° C. and 10° C., respectively (FIG. 9D). Interestingly, theunfolding of m02 was broader and with lower peak than those of CH2 andm01. This phenomenon could be caused by the presence of dimers in m02.

The stability against chemically induced unfolding of m01 and m02 wasalso higher than that of CH2 (FIG. 9E). Urea was used as a chemicalagent to measure the intrinsic fluorescence spectra. The unfoldingdependences on the urea concentration can be also fitted by a two-statemodel. The 50% unfolding of m01 and m02 occurred at higher ureaconcentrations (6.8 and 5.8 M, respectively) than that of CH2 (4.2 M).

Only monomer fraction was observed for m01 while m02 contained smallamounts of higher molecular species, mostly dimers as determined by SEC(FIG. 10). Because of its superior properties, m01 was selected forfurther characterization. the stability of a truncated CH2 (CH2s) and atruncated m01 (mOls) where the first seven N-terminal residues weredeleted (residues 1-7 of SEQ ID NO: 5) were also tested. These truncatedproteins exhibited high stability. The 50% unfolding temperatures (Tms)measured by CD (62° C. and 79° C., respectively) are significantlyhigher (8° C. and 5° C., respectively) than those of the correspondingCH2 and m01 (54° C. and 74° C., respectively) (FIG. 10B).

Structural Conservation of m01.

To examine structural perturbation caused by the cysteine mutations,solution NMR experiments were performed for the CH2 domain and the m01mutant. ¹H-¹⁵N HSQC spectrum generally shows a correlation of nitrogenatoms and their directly bounded protons, and provides a “fingerprint”of the protein backbone. Each of the ¹H-¹⁵N HSQC spectra of CH2 and them01 (recorded in identical experimental conditions) exhibited only oneset of peaks, indicating that the protein was well-folded in solution.Of the structure region of the proteins, the chemical shifts of backbone¹⁵N, C′, and C_(α) were ca. 75% assigned in both proteins. In m01, themeasured chemical shifts for C_(α)and C_(β) of residue Cys 12 were 57.6ppm and 37.7 ppm, respectively, whereas the C_(α) and C_(β) chemicalshifts of Cys 104 were 34.2 ppm and 54.5 ppm, respectively. These valuesfall within the expected range for oxidized cysteine residues (Sharmaand Rajarathnam, J. Biomol. NMR 18:165-171, 2000), demonstrating thatthe additional disulfide bridge is formed in the m01 mutant.

Comparison of the overall backbone chemical shifts of N and C_(α) alsoshowed the overall similarity of the protein structures between CH2 andthe m01. However, changes in chemical shifts were observed aroundresidues Cys31 and Cys91 as well as around the newly introduced Cysresidues 12 and 104. This is not unexpected because the newly introduceddisulfide bridge is proximal to the native Cys31-Cys91 by linking theadjacent β-strands in the same β-sheet with the Cys31-Cys91 bridge. Thenewly introduced disulfide bond in CH2 m01 most likely affectedmicroscopic environments of the native disulfide bond between Cys31 andCys91.

Relatively High Loop Flexibilities and Rigid Framework of CH2 and m01.

To determine whether the loops are flexible in both CH2 and m01,¹⁵N-{¹H} NOE was recorded. It was determined that the framework is rigidas indicated by the high NOE values (above 0.7); in contrast the loopswere on average more flexible. The local dynamics of CH2 and m01 werecomparable, demonstrating that the conformational entropy of m01 at thenative states is very similar to that of CH2. It is most likely that theessential structure and dynamics of the CH2 domain is maintained whilethermal stability is increased upon introduction of the cysteinemutation. The increase in the flexibility of the loops also indicatesthat both CH2 and m01 could be used as scaffolds for grafting to ormutating residues in the loops.

Example 4 CH2 Domain Molecules Specific for HIV

This example describes the construction of a synthetic phage library,based on the loops of the CH2 domain of human IgG1, to identify CH2molecules that specifically bind HIV envelope.

Materials and Methods

Primers, Peptide and Proteins.

All the primers used in this study were purchased from Invitrogen(Carlsbad, Calif.). The biotin labeled peptide was from Sigma (St.Louis, Mo.). Bal gp120-CD4 was kindly provided by Tim Fouts (Universityof Maryland, Baltimore, Md.) and other gp120/140 were provided byChristopher Broder (USUHS, Bethesda, Md.). SCD4 was obtained throughAIDS research and reagent program.

Library Construction.

Overlapping PCR was used to introduce mutations to loops 1 and 3 togenerate the first CH2 based library. N terminus primer ACGT GGCC CAGGCGGCC GCA CCT GAA CTC CTG (SEQ ID NO: 101) and loop 1 primer CAC GTA CCAGTT GAA CTT GCC AKM AKM AKM AKM AKM AKM AKM AKM AKM AKM CAC CAC CAC GCATGT GAC (SEQ ID NO: 7) were used to generate the N terminal half of theCH2 containing mutations in loop 1. Loop 1 linkage primer AAG TTC AACTGG TAC GTG (SEQ ID NO: 8) and loop 3 primer GAT GGT TTT CTC GAT GGG GCCAKM AKM AKM AKM AKM AKM GTT GGA GAC CTT GCA CTT G (SEQ ID NO: 9) wereused to generate the rest of CH2 with mutations in loop 3. The twofragments were then combined by an overlapping PCR step and amplifiedwith the N terminus primer and C terminus primer ACGT GGCC GGCCT GGCCTTT GGC TTT GGA GAT GGT TTT CTC GAT G (SEQ ID NO: 102) with a SfiI site(underlined) being introduced into both ends of the CH2 fragment. Togenerate the secondary library based on the binders isolated from thefirst library, loop 2 primer GCT GAC CAC ACG GTA ADH ADH ADH GTA CTG CTCCTC CCG (SEQ ID NO: 103) and above described N terminus primer were usedto introduce mutations to loop 2 to the primary binder. Loop 2 linkageprimer TAC CGT GTG GTC AGC (SEQ ID NO: 104) and loop 3 primer (2) GGAGAT GGT TTT CTC GAT GGG ADH TGG ADH ADH ADH GTT GGA GAC CTT GCA (SEQ IDNO: 105) were used to introduce mutations to the primary binder. The twofragments were joined by an overlapping PCR step and amplified using thesame pair of N and C terminus primers described above for amplification.PCR fragments were subject to SfiI digestion and ligated to the vector.The ligated product was desalted and transformed to theelectro-competent TG1 cells suing an electroporator (Bio-Rad, Hercules,Calif.). A phage library was prepared from the resulted transformants.

Panning.

Bal gp120-CD4, Bal gp120 as well as BSA were coated directly to Maxisorpplates (Nunc, Denmark) in PBS buffer at 4° C., overnight for a plateformat panning. Approximately 10¹³ phage particles of the respective CH2libraries were suspended in PBS with 2% dry milk and applied to wellscoated with the proteins. After 2 hours at room temperature, each wellwas washed 5 times for the first round and 10 times for the subsequentrounds before the phages were rescued with TG1 cells at the exponentialgrowth phase. A total of five rounds of panning were performed for eachantigen for the first library. For the second library based on theprimary binder, three rounds of panning were performed. Monoclonal ELISAwas then used to select for positive clones. Two hundred clones werescreened for each antigen. Only clones displaying an OD 405>2.0 in themonoclonal ELISA were selected for plasmid preparation and sequencing.

CH2 Expression and Refolding.

Clones selected as described above were transformed into E. coli strainHB2151 for expression. Briefly, a single clone was inoculated into 2×YTsupplemented with 100 units of amp and incubated at 37° C. with shaking.When the OD₆₀₀ reached 0.5, IPTG was added to achieve a finalconcentration of 1 mM and the culture was continued with shaking foranother 3-5 hours. Cells were then collected, lysed with polymyxin B(Sigma, St Louis) in PBS, and the supernatant was subjected to Ni-NTAagarose bead (Qiagen, Hilden, Germany) purification for the solubleportion of the CH2 clones. The pellet was then re-suspended in buffercontaining 25 mM Tris.HCl, pH 8.0, 6 M Urea, 0.5 M NaCl, and subjectedto brief sonication. The supernatant was collected by centrifugation andsubjected to Ni-NTA agarose bead (Qiagen) purification. CH2 obtainedthrough the pellet was subjected to overnight dialysis against twochanges of PBS and then filtered through a 0.2 μm low protein bindingfilter (Pal, Ann Arbor, Mich.).

ELISA.

Different protein antigens were diluted in the PBS buffer inconcentrations ranging from 1-4 μg/ml and coated to the 96 well plate at4° C. overnight. The plate was then blocked with PBS+5% dry milk buffer.CH2 clones in different concentrations were diluted in the same blockingbuffer and applied to the ELISA plate. Mouse-anti-His-HRP was used todetect the His tag at the C terminal end of each of the CH2 clones inmost of the ELISA unless indicated otherwise. ABTS was then added toeach well and OD₄₀₅ was taken 5-10 minutes afterward.

Gel Filtration Analysis.

Samples of purified and filtered CH2 proteins were analyzed on aSuperdex75 10/300GL column (GE Healthcare, Piscataway, N.J.)pre-equilibrated with PBS. The column was calibrated with molecularweight standards. CH2 samples were eluted from the column at a flow rateof 0.5 ml/min.

Pseudovirus Neutralization Assay.

HIV Env pseudotyped virus preparation and neutralization was performedessentially as previously described (Choudhry et al., Virology363:79-90, 2007).

Results

Design and Construction of a Human CH2-Based Library.

It was hypothesized that limited mutagenesis of the CH2 loops may notsignificantly affect the folding and stability of many mutants and couldbe used for the generation of large libraries of potential binders.First, mutagenesis of loop 1 (L1) and loop 3 (L3) was undertaken becausethey are the longest (9 and 5 residues, respectively) two loops on thesame side of the molecule (loops BC, DE and FG are herein referred to asL1, L2 and L3, respectively; the two helices AB and EF are referred toas H1 and H2, respectively; and the loop CD is referred to as L0) (FIG.11) (Radaev et al., J. Biol. Chem. 276:16469-16477, 2001). Fourfrequently occurring residues in CDRs (A, Y, D, and S) were selected torandomly replace all L1 and L3 residues and to add one additionalresidue. An additional residue (G) was also added to the C-terminal endof each loop to increase flexibility (FIG. 11). It has been previouslyobserved that these four residues (sometimes only two) are sufficient tobuild a specific binding surface within different frameworks (Fellouseet al., Proc. Natl. Acad. Sci. USA 101:12467-12472, 2004; Koide et al.,Proc. Natl. Acad. Sci. USA 104:6632-6637, 2007). The calculatedtheoretical diversity of this library is 4¹⁶=4.3×10⁹. However, due topotential mutations generated by PCR (see below) the diversity is likelyto be significantly higher up to the size of the library (5×10¹⁰). Mostmutants (probably greater than 80%) have correct reading frames asindicated by an analysis of 100 randomly selected clones.

Identification and Sequence Analysis of Binders.

To test the library and select potentially useful binders, an HIV-1envelope glycoprotein, gp120, from the Bal isolate, fused with atwo-domain CD4 (denoted as gp120_(Bal)-CD4) was used as an antigen.After five rounds of panning, 200 clones were screened by phage ELISAand 15 clones with the highest signal were isolated for furthercharacterization. Three clones, m1a1, m1a2 and m1a3, dominatedrepresented by 7, 5 and 2 (out of 15) sequences, respectively,suggesting a specific enrichment. They have similar L1 sequences,composed mostly of D and Y but their L3s are very different. The mostabundant clones, m1a1 and m1a2, have several changes in L1 (two Fs inL1, and deletion before G, respectively) apparently due to PCR errors.The loop 1 and loop 3 sequences of the clones selected against Balgp120-CD4 are shown below in Table 8. These results suggest thatCH2-based scaffolds can support phage-displayed binders with varying L1and L3; the newly identified HIV-1-specific binders were furthercharacterized as described below.

TABLE 8 CH2 Loop 1 and Loop 3 Sequences Loop 1 Loop 3 SEQ Clone sequenceSEQ ID NO: sequence ID NO: m1a1 DYDYDSYFDFG 107 SDSAASG 110 m1a2DYDYDSYYD..G 108 DDYAADG 111 m1a3 DYDYDSYYDYG 109 YDYADDG 112 m1a3'*DYDYDSYYDYG 109 SDYDSSG 113 wt CH2 DVSHEDPEV  93 KALPA  95 (aa 4-12)(aa 4-8) *The m1a3′ clone has the same loop 1 sequence as m1a3 but has adifferent loop 3 sequence

Expression Of Soluble nAbs and Characterization of their Binding.

Most of the expressed CH2 domain molecules (referred to as “nAbs”) werefound in inclusion bodies (FIG. 12A) and were refolded as describedabove, yielding on average 10-30 mg per L of bacterial culture. Thepurified nAbs bound to the panning antigen (gp120-CD4) specifically asmeasured by ELISA with EC50s ranging from 500 nM (m1a1 and m1a2) to lowμM (m1a3) (FIG. 12B). Similar results were obtained for nAbs purifiedfrom the supernatant. These results suggest that m1a1, m1a2 and m1a3retain their binding activity in soluble (not phage-displayed) form andthat refolding from inclusion bodies does not affect these molecules.The two clones with highest affinity, m1a1 and m1a2, were selected forfurther characterization.

To test their cross-reactivity, four (Bal, JRFL, R2 and 89.6)recombinant HIV-1 envelope glycoproteins were used alone and in complexwith soluble CD4. As shown in FIG. 14, m1a1 binds to various degrees toall proteins. While m1a1 binds to Bal gp120 in complex with CD4, butvery weakly to gp120 alone as expected for a CD4 induced (CD4i)antibody, its binding to the other proteins was not affectedsignificantly by the presence of CD4. The decrease in signal for the Envalone is not significant and could be due to the slightly reducedcoating by gp120 when mixed with sCD4. Similar results were obtained form1a2. These data suggest that the epitope recognized by these antibodiesis CD4i for one isolate (Bal) but not for the others.

To further characterize their epitope, m1a1 competition with alreadyknown CD4i antibodies (scFv X5 and the domain antibody m36) was tested.Both CD4i antibodies competed significantly with m1a1. Therefore, m1a1recognizes a novel conserved epitope that is shared by other highlypotent cross-reactive CD4i antibodies, but in contrast to thoseantibodies its exposure by the gp120 interaction with CD4 issignificantly dependent on the isolate.

Loop 1 Determines the Binding Specificity.

To determine the different contributions of the loops from the CH2clones to the specific binding, two hybrid clones were generated:m1a1CH2 and m1a2CH2. L1s from m1a1 and m1a2 were grafted onto CH2replacing the original L1. These hybrid antibodies bound to gp120-CD4with about the same although slightly lower affinity compared to m1a1 asmeasured by ELISA (FIG. 13A), indicating that L3s are not essential forbinding. To find out whether the scaffold as a whole is required forbinding, m1a1 L1 was tested in isolation as a synthetic peptide(DYDYDSYFDFG; SEQ ID NO: 109). The biotin labeled peptide did not bind.The effect of relatively minor conformational changes in the scaffold onbinding was also tested by creating an additional disulfide bond betweenstrands A and G. As described in Example 3, such S—S bond increasessignificantly the CH2 stability and does not affect significantly themobility and the microenvironment of any CH2 residue as measured by NMR.The resulting antibody m1a1ss did not bind either (FIG. 13B). These datasuggest that the scaffold is required for the binding activity of m1a1,and that while changes in L3 may not affect its activity, relativelysmall changes in the scaffold conformation could abolish it.

Neutralization of HIV-1 Pseudovirus by m1a1 and m1a2.

To assess the neutralizing activity of m1a1 and m1a2, a cellline/pseudovirus assay and a panel of nine HIV-1 isolates was used.Seven of these isolates were inhibited to a certain degree by one orboth antibodies (FIG. 14A). The two antibodies differentially inhibitedtwo isolates (89.6 and IIIB) and to about the same degree five otherisolates (FIG. 14A). As expected from their relatively modest bindingaffinity, their potency was relatively modest compared to the highlypotent inhibitor C34 used here as positive control. These resultsprovide proof of concept that functional binders can be selected fromlibraries based on the CH2 scaffold.

The antibodies were further improved by mutagenesis of the second andthird loop (FIG. 15). They ran mostly monomeric on SDS gels (FIG. 16A).One of the mutants, m1b3, was mostly monomeric in gel filtration (FIG.16B). They bound specifically (FIG. 16C) and neutralize to variousextent HIV-1 (FIG. 17). They also competed with scFv X5 and m36suggesting that they target a highly conserved region on the HIV-1 gp120(FIG. 18).

This disclosure provides antibody constant domain molecules comprisingat least one mutation, or at least one CDR, or functional fragmentthereof. The disclosure further provides compositions comprising theantibody constant domain molecules and their use. It will be apparentthat the precise details of the methods described may be varied ormodified without departing from the spirit of the described invention.We claim all such modifications and variations that fall within thescope and spirit of the claims below.

1. A nucleic acid molecule encoding a polypeptide comprising animmunoglobulin CH2 domain of IgG, IgA or IgD, or a CH3 domain of IgE orIgM, wherein the CH2 domain or CH3 domain comprises an N-terminaltruncation of about 1 to about 7 amino acids, and wherein (i) at leastone of the loops of the CH2 or CH3 domain is mutated; (ii) at least aportion of a loop region of the CH2 domain or CH3 domain is replaced bya complementarity determining region (CDR), or a functional fragmentthereof, from a heterologous immunoglobulin variable domain; or (iii)both, wherein the polypeptide has a molecular weight of less than about15 kD, and wherein the polypeptide specifically binds an antigen.
 2. Avector comprising the nucleic acid molecule of claim
 1. 3. An isolatedhost cell comprising the vector of claim
 2. 4. A method of treating HIV,cancer or an autoimmune or inflammatory disorder in a subject,comprising administering to the subject a therapeutically effectiveamount of a polypeptide comprising an immunoglobulin CH2 domain of IgG,IgA or IgD, or a CH3 domain of IgE or IgM, wherein the CH2 domain or CH3domain comprises an N-terminal truncation of about 1 to about 7 aminoacids, and wherein (i) at least one of the loops of the CH2 or CH3domain is mutated; (ii) at least a portion of a loop region of the CH2domain or CH3 domain is replaced by a complementarity determining region(CDR), or a functional fragment thereof, from a heterologousimmunoglobulin variable domain; or (iii) both, wherein the polypeptidehas a molecular weight of less than about 15 kD, and wherein thepolypeptide specifically binds an antigen from HIV, a cancer-specificantigen, a cancer-related protein, or an antigen related to anautoimmune or inflammatory disorder.
 5. The method of claim 4, whereinthe method is a method of treating cancer and the cancer is leukemia,lymphoma, multiple myeloma, malignant melanoma, breast cancer, lungcancer, prostate cancer, colon cancer or renal cell carcinoma.
 6. Apolypeptide comprising an immunoglobulin CH2 domain of IgG, IgA or IgD,or a CH3 domain of IgE or IgM, wherein the CH2 domain or CH3 domaincomprises a first amino acid substitution and a second amino acidsubstitution, wherein the first and second amino acid substitutions eachreplace the original residue with a cysteine residue, wherein thecysteine residues form a disulfide bond, and wherein the polypeptide hasa molecular weight of less than about 15 kD.
 7. The polypeptide of claim6, wherein the first amino acid substitution is in the N-terminal Astrand and the second amino acid substitution is in the C-terminal Gstrand.
 8. The polypeptide of claim 6 comprising a CH2 domain of IgG. 9.The polypeptide of claim 8, wherein the first amino acid substitution is(i) L12 to C12 or (ii) V10 to C10, and the second amino acidsubstitution is K104 to C104 (numbered with reference to SEQ ID NO: 5).10. The polypeptide of claim 6, wherein the CH2 or CH3 domain comprisesan N-terminal truncation of about 1 to about 7 amino acids.
 11. Thepolypeptide of claim 6, wherein (i) at least one of the loops of the CH2or CH3 domain is mutated; (ii) at least a portion of a loop region ofthe CH2 domain or CH3 domain is replaced by a complementaritydetermining region (CDR), or a functional fragment thereof, from aheterologous immunoglobulin variable domain; or (iii) both, and whereinthe polypeptide specifically binds an antigen.
 12. A polypeptidecomprising an immunoglobulin CH2 domain of IgG, IgA or IgD, or a CH3domain of IgE or IgM, wherein the CH2 domain or CH3 domain comprises anN-terminal truncation of about 1 to about 7 amino acids, and wherein thepolypeptide has a molecular weight of less than about 15 kD.
 13. Thepolypeptide of claim 12, comprising an N-terminal truncation of about 7amino acids.
 14. The polypeptide of claim 12, wherein the CH2 domain orCH3 domain further comprises a first amino acid substitution and asecond amino acid substitution, wherein the first and second amino acidsubstitutions each replace the original residue with a cysteine residue,wherein the cysteine residues form a disulfide bond.
 15. The polypeptideof claim 12, wherein (i) at least one of the loops of the CH2 or CH3domain is mutated; (ii) at least a portion of a loop region of the CH2domain or CH3 domain is replaced by a complementarity determining region(CDR), or a functional fragment thereof, from a heterologousimmunoglobulin variable domain; or (iii) both, and wherein thepolypeptide specifically binds an antigen.
 16. A method of identifying arecombinant CH2 domain or CH3 domain that specifically binds a targetantigen, comprising: (a) providing a library of particles displaying ontheir surface a recombinant CH2 or CH3 domain, wherein the CH2 or CH3domain comprises an N-terminal deletion of about 1 to about 8 aminoacids, and wherein the CH2 or CH3 domain has a molecular weight lessthan about 15 kD, wherein the library is generated by: (i) providing alibrary of nucleic acid molecules encoding a genetically diversepopulation of CH2 or CH3 domains, wherein the genetically diversepopulation is provided by introducing mutations into one or more loopregions of the CH2 or CH3 domain; and (ii) expressing the library ofnucleic acid molecules in recombinant host cells, whereby the CH2domains or CH3 domains are expressed on the surface of the particles andthe CH2 or CH3 domain nucleic acid molecules are encoded by the geneticmaterial of the particles; (b) contacting the library of particles withthe target antigen to select particles that specifically bind the targetantigen; and (c) cloning the CH2 or CH3 domain nucleic acid moleculesfrom the particles expressing the CH2 or CH3 domains that specificallybind the target antigen, thereby identifying a CH2 or CH3 domain thatspecifically binds the target antigen.
 17. The method of claim 16,wherein the CH2 domain or CH3 domain comprises at least one mutation inLoop 1, at least one mutation in Loop 2, at least one mutation in Loop3, at least one mutation in Loop A-B, at least one mutation in Loop C-D,or at least one mutation in E-F, or any combination thereof.
 18. Themethod of claim 16, wherein the library comprises recombinant CH2domains.
 19. The method of claim 18, wherein the CH2 domain is an IgGCH2 domain.
 20. The method of claim 16, wherein the particles are phageparticles.
 21. A method of making a library of recombinant CH2 or CH3domains, comprising (i) introducing mutations into one or more loopregions of a CH2 domain or CH3 domain scaffold, or (ii) replacing aportion of a loop region of the CH2 domain or CH3 domain scaffold with aCDR or functional fragment thereof from a heterologous immunoglobulinvariable domain, or (iii) both, wherein the scaffold comprises anisolated immunoglobulin CH2 domain of IgG, IgA or IgD or CH3 domain ofIgE or IgM.
 22. The method of claim 21, wherein the CH2 or CH3 domainscaffold further comprises an N-terminal truncation of about 1 to about7 amino acids.
 23. The method of claim 21, wherein the CH2 or CH3 domainscaffold further comprises a C-terminal truncation of about 1 to about 4amino acids.
 24. The method of claim 21, wherein the CH2 or CH3 domainscaffold further comprises a first amino acid substitution and a secondamino acid substitution, wherein the first and second amino acidsubstitutions each replace the original residue with a cysteine residue,wherein the cysteine residues form a disulfide bond.
 25. A method ofidentifying a recombinant CH2 domain or CH3 domain that specificallybinds a target antigen, comprising contacting the library produced bythe method of claim 21 with the target antigen to select recombinant CH2or CH3 domains that specifically bind the target antigen.